BIOLOGY 

R 

G 


ANTONI   VAN   LEEUWENHOEK 
1682-1723 


THE  PRINCIPLES 


OF 


BACTEEIOLOGT 


A  PRACTICAL  MANUAL  FOR  STUDENTS 
AND  PHYSICIANS 


BY 


A.  C.  ABBOTT,  M.D. 


PROFESSOR  OF  HYGIENE  AND  BACTERIOLOGY,  AND  DIRECTOR  OF  THE    SCHOOL    OF 
HYGIENE  AND  PUBLIC  HEALTH,  UNIVERSITY  OF    PENNSYLVANIA 


TENTH  EDITION,  THOROUGHLY  REVISED 


WITH  121   ILLUSTRATIONS,  31  OF  WHICH  ARE  COLORED 


LEA   &   FEBIGER 

PHILADELPHIA  AND   NEW  YORK 


BIOLOGY 

LIBRARY 

G 


COPYRIGHT 

LEA  &  FEBIGER 

1921 


PRINTED  IN  U.  S.  A. 


PREFACE  TO  THE  TENTH  EDITION. 


As  in  previous  editions,  we  have  kept  before  us  the  adapta- 
tion of  this  book  to  the  needs  of  the  beginner.  There  have 
been  incorporated  all  advances  that  we  regard  as  fundamental 
to  an  understanding  of  the  accepted  principles  and  practices 
of  modern  bacteriology. 

Opinion  is  now  well  stabilized  upon  the  matter  of  variations 
in  bacterial  species  and  the  subject  has  been  treated  in  appro- 
priate places  in  both  a  general  and  particular  manner. 

Since  the  last  edition  of  this  book  there  have  been  devel- 
oped methods  for  the  estimation  of  the  exact  acid  or  alkaline 
strengths  of  solutions.  These  methods  are  based  upon  the 
theory  of  dissociation  of  electrolytes  under  different  condi- 
tions of  solution.  They  are  now  so  simplified  as  to  make 
them  adaptable  to  the  routine  estimation  and  correction  of 
reactions  of  culture  media.  In  this  connection  an  effort  has 
been  made  to  explain  the  phenomenon  of  dissociation  in  such 
manner  as  to  make  it  comprehensible  to  those  not  versed  in 
the  physics  of  solutions,  and  to  clarify  the  steps,  and  reasons 
therefor,  taken  in  efforts  to  determine  the  reactions  of 
solutions,  especially  of  culture  media,  by  the  estimation  of 
their  hydrogen-ion  content.  The  significance  of  pH  values 
as  symbols  of  reaction  and  the  use  of  indicators  for  different 
pH  values  is  explained. 

689G30 


iv  PREFACE  TO  THE  TENTH  EDITION 

Recently  work  upon  that  ill-defined  group  of  micro- 
organisms, the  Spirochsetaceae,  has  at  least  begun  to  be 
systematized,  and  already  our  knowledge  of  their  nature, 
their  relation  to  disease  and  the  possible  distribution  of  this 
group  have  been  greatly  extended.  Sufficient  of  these 
studies  has  been  incorporated  in  this  edition  to  give  to  the 
student  an  idea  of  what  has  been  accomplished  and  the 
methods  used.  New  illustrations  accompany  this  section. 

Since  nothing  has  transpired  to  shake  the  foundations  of 
the  science  of  bacteriology,  the  fundamental  features  of  the 
book,  except  for  minor  changes,  remain  as  in  the  last  edition. 

A.  C.  A. 

UNIVERSITY  OP  PENNSYLVANIA, 
1921. 


CONTENTS. 


INTRODUCTION. 

"Omne  Vivum  ex  Vivo"— The  Overthrow  of  the  Doctrine  of 
Spontaneous  Generation — Earlier  Bacteriological  Studies — 
The  Birth  of  Modern  Bacteriology 17 

CHAPTER  I. 

Definition  of  Bacteria — Differences  Between  Parasites  and 
Saprophytes — Their  Place  in  Nature — Bacterial  Enzymes 
— Products  of  Bacteria — Nutrition  of  Bacteria — Their  Rela- 
tion to  Oxygen — Influence  of  Temperature  Upon  Their 
Growth — Chemotaxis 33 

CHAPTER  II. 

Morphology  of  Bacteria — Chemical  Composition  of  Bacteria — 

Mode  of  Multiplication — Spore-formation — Motility        .      .       63 

CHAPTER  III. 

^Principles  of  Sterilization  by  Heat— Methods  Employed — Dis- 
continued Sterilization — Fractional  Sterilization — Apparatus 
Employed — Sterilization  under  Pressure — Sterilization  by 
Hot  Air — Thermal  Death-point  of  Bacteria — Chemical  Dis- 
infection and  Sterilization — Mode  of  Action  of  Disinfectants 
— Practical  Disinfection 76 

CHAPTER  IV. 

Principles  Involved  in  the  Methods  of  Isolation  of  Bacteria  in 
Pure  Culture  by  the  Plate  Method  of  Koch — Materials 
Employed 104 

(v) 


vi  CONTENTS 


CHAPTER  V. 

Reactions,  Methods  for  Adjustment — Titration — Hydrogen  ion 
Concentration — Preparation  of  Media — Bouillon,  Gelatin, 
Agar-agar,  Potato,  Blood  Serum,  Blood  Serum  from  Small 
Animals,  Milk,  Litmus-whey  Milk,  Dm  ham's  Peptone  Solu- 
tion, Lactose  Litmus-agar,  Loffler's  Blood  Serum  Mixture, 
the  Serum  Water  Media  of  Hiss,  Guarniari's  Gelatin-agar 
Mixture Ill 

CHAPTER  VI. 

Preparation  of  the  Tubes,  Flasks,  etc.,  in  which  the  Media  are  to 

be  Preserved 145 

CHAPTER   VII. 
Technique  of  Isolating  Bacteria  in  Pure  Culture  by  the  Plate  and 

the  Tube  Method 149 

CHAPTER  VIII. 

The  Incubating  Oven — The  Safety  Burner  Employed  in  Heating 

the   Incubator — Thermo-regulator — Gas-pressure   Regulator     157 

CHAPTER  IX. 

The  Study  of  Colonies — Their  Naked-eye  Peculiarities  and  Their 
Appearance  Under  Different  Conditions — Differences  in  the 
Structure  of  Colonies  from  Different  Species  of  Bacteria — 
Stab  Cultures— Slant  Cultures 165 

CHAPTER  X. 

Methods  of  Staining — Cover-slip  Preparations — Impression 
Cover-slip  Preparations — Solutions  Employed — Preparation 
and  Staining  of  Cover-slips — Staining  Solutions — Special 
Staining  Methods 171 

CHAPTER  XL 

Systematic  Study  of  an  Organism — Points  to  be  Considered  in 
Determining  the  Morphologic  and  Biologic  Characters  of  a 
Culture — Methods  by  which  the  Various  Biologic  and  Chem- 
ical Characters  of  a  Culture  may  be  Ascertained — Variation 
and  Varieties — Dark  Field  Illumination — Facts  Necessary 
to  Permit  the  Identification  of  an  Organism  as  a  Definite 
Species 191 


CONTENTS  vn 


CHAPTER  XII. 

Inoculation  of  Animals — Subcutaneous  Inoculation — Intravenous 
Injection — Inoculation  into  the  Lymphatic  Circulation — 
Inoculation  into  the  Great  Serous  Cavities  and  into  the 
Anterior  Chamber  of  the  Eye — Observation  of  Animals 
after  Inoculation  .  232 


CHAPTER  XIII. 

Post-mortem  Examination  of  Animals — Bacteriological  Examina- 
tion of  the  Tissues — Disposal  of  Tissues  and  Disinfection  of 
Instruments  after  the  Examination — Study  of  Tissues  and 
Exudates  During  Life 255 

CHAPTER  XIV. 

Infection  and  Immunity — Mechanism — Defenses  of  the  Body — 

Specific  Bodies  and  Reactions — Doctrines  in  Explanation      .     266 

CHAPTER  XV. 

Hemolysis — The  Hemolytic  System — Identification  of  Specific 
Immune  Bodies  and  Specific  Antigens  by  their  Ability  to 
Fix  Complement — The  Wassermann  Reaction — Schematic 
Representation  of  Reactions 312 


APPLICATION  OF  THE  METHODS  OF 
BACTERIOLOGY. 


CHAPTER  XVI. 
To  Obtain  Material  with  Which  to  Begin  Work 323 

CHAPTER  XVII. 
Various  Experiments  in  Sterilization  by  Steam  and  by  Hot  Air    .     327 


viii  CONTENTS 


CHAPTER  XVIII. 

Methods  of  Testing  Disinfectants  and  Antiseptics — Experiments 
Illustrating  the  Precautions  to  be  Taken — Experiments  in 
Skin-disinfection 331 

CHAPTER  XIX. 

Micrococcus  Aureus — Micrococcus  Pyogenes  and  Citreus — 
Staphylococcus  Epidermidis  Albus — Streptococcus  Pyogenes, 
Types  and  Typing — Micrococcus  Gonorrhcese — Micrococcus 
Intracellularis,  Varieties  —  Pseudomonas  ^Eruginosa  — 
Bacillus  of  Bubonic  Plague,  Protective  Inoculation  and  Anti- 
serum  345 

CHAPTER  XX. 

Some  of  the  Pathogenic  Organisms  Encountered  in  the  Mouth 
Cavity  in  Health  and  Disease — Micrococcus  Lanceolatus, 
(Pneumococcus)  Types  and  Typing — Micrococcus  Tetrag- 
enus,  Bacterium  Influenzae,  Bacillus  Tuberculosis,  etc.  .  .  401 

CHAPTER  XXI. 

Tuberculosis — Microscopic  Appearance  of  Miliary  Tubercles — 
Diffuse  Caseation — Cavity-formation — Encapsulation  of 
Tuberculous  Foci — Primary  Infection — Modes  of  Infection, 
Vaccination  Against — The  Bacterium  Tuberculosis — Loca- 
tion of  the  Bacilli  in  the  Tissues — Microscopic  Appearance  of 
Bacterium  Tuberculosis — Staining  Peculiarities — Organisms 
with  Which  Bacterium  Tuberculosis  may  be  Confounded: 
Bacterium  Leprse;  Bacterium  Smegmatis — Acid-proof  Bac- 
teria— Bacterium  Tuberculosis  Avium — Variations — Pseudo- 
tuberculosis — Susceptibility  of  Animals — Tuberculin — Vac- 
cination Against  Tuberculosis — Actinomyces  Bovis — Actino- 
myces  Israeli,  Actinomyces  Madurse,  Actinomyces  Farcinicus, 
Actinomyces  Eppingeri,  Actinomyces  Pseudo  tuberculosis  .  430 

CHAPTER  XXII. 

Glanders— Characteristics  of  the  Disease — Histological  Structure 
of  the  Glanders  Nodule — Susceptibility  of  Different  Animals 
to  Glanders — The  Bacterium  of  Glanders;  Its  Morpholog- 
ical and  Cultural  Peculiarities — Diagnosis  of  Glanders — 
Mallein  .  470 


CONTENTS  IX 


CHAPTER  XXIII. 

Bacterium  (Syn.  Bacillus)  Diphtherise — Its  Isolation  and  Cultiva- 
tion—Morphological and  Cultural  Peculiarities— Pathogenic 
Properties — Variations  in  Virulence — Varieties — Bacterium 
Pseudodiphtheriticum  —  Bacterium  Xerosis  —  Diphtheria 
Antitoxin 480 

CHAPTER  XXIV. 

Typhoid  Fever — Study  of  the  Organism  Concerned  in  its  Produc- 
tion— Its  Morphological,  Cultural,  and  Pathogenic  Properties 
— Prophylactic  Inoculations — Bacillus  Coli — Bacillus  Para- 
typhosus — Its  Resemblance  to  Bacillus  Typhosus  .  .  .  508 

CHAPTER  XXV. 

The  Group  of  Bacilli  Found  in  Cases  of  Epidemic,  Endemic,  and 
Sporadic  Dysentery — The  Morphological,  Biological,  and 
Pathogenic  Characters  of  the  Several  Members  of  the  Group 
—The  Differentiation  of  the  Different  Types  of  Bacilli  .  .541 

CHAPTER  XXVI. 

The  Spirillum  (Comma  Bacillus)  of  Asiatic  Cholera — Its  Mor- 
phological and  Cultural  Peculiarities — Pathogenic  Properties 
— The  Bacteriological  Diagnosis  of  Asiatic  Cholera — Micro- 
spira  Metchnikovi — Microspira  ("Vibrio")  Schuylkilliensis 
— Its  Morphological,  Cultural,  and  Pathogenic  Characters  .  549 

CHAPTER  XXVII. 

Study  of  Bacterium  Anthracis,  and  of  the  Effects  Produced  by  Its 
Inoculation  into  Animals — Peculiarities  of  the  Organism 
Under  Varying  Conditions  of  Surroundings — Anthrax  Vac- 
cines— Anthrax  Immune  Serum 583 

CHAPTER  XXVIII. 

The  Nitrifying  Bacteria— The  Bacillus  of  Tetanus— The  Bacillus 
of  Malignant  Edema — The  Bacillus  of  Symptomatic  Anthrax 
— Bacterium  Welchii — Bacillus  Sporogenes — The  Spirochae- 
tacese,  Spironema,  Treponema,  Leptospira  —  Relapsing 
Fever,  Yellow  Fever,  Syphilis 600 


CONTENTS 


CHAPTER  XXIX. 

Bacteriological  Study  of  Water — Methods  Employed — Precau- 
tions to  be  Observed — Apparatus  Employed,  and  Methods  of 
Using  It — Methods  of  Investigating  Air  and  Soil — Bacterio- 
logical Study  of  Milk — Methods  Employed  .  .  .  .  .  637 


APPENDIX  669 


BACTERIOLOGY. 


INTRODUCTION:',  >,„  "        ; .- '; } ; , ;  ; 

"Omne  Vivum  ex  Vivo" — The  Overthrow  of  the  Doctrine  of  Spontaneous 
Generation — Earlier  Bacteriological  Studies — The  Birth  of  Modern 
Bacteriology. 

BACTERIOLOGY  may  be  said  to  have  had  its  beginning 
with  the  observations  -of  Leeuwenhoek  in  the  latter  part 
of  the  seventeenth  century.  Though  its  most  rapid  and 
important  development  has  taken  place  since  about  1880, 
still,  a  review  of  the  various  evolutionary  phases  through 
which  it  has  passed  in  the  course  of  more  than  two  hundred 
years  reveals  an  entertaining  and  instructive  history.  From 
the  very  outset  its  history  is  inseparably  connected  with 
that  of  medicine,  and  from  the  outcome  of  bacteriological 
research  preventive  medicine,  in  its  modern  conception, 
received  its  primary  impulse.  Through  a  more  intimate 
acquaintance  with  the  biological  activities  of  the  unicellular 
vegetable  microorganisms  modern  hygiene  has  attained 
almost  the  dignity  of  an  exact  science,  and  properly  merits 
the  importance  and  prominence  now  generally  accorded  to 
it.  From  studies  in  the  domain  of  bacteriology  our  knowl- 
edge of  the  causation,  course,  and  prevention  of  infectious 
diseases  is  daily,  becoming  more  accurate,  and  it  is  needless 
to  emphasize  the  relation  of  such  knowledge  to  the  manifold 
problems  that  present  themselves  to  the  student  of  modern 
2  (17) 


18  BACTERIOLOGY 

medicine.  Though  the  contributions  which  have  done  most 
to  place  bacteriology  on  the  footing  of  a  science  are  those 
of  recent  years,  still,  during  the  earlier  stages  of  its  devel- 
opment, many  observations  were  made  which  formed  the 
foundation-work  for  much  that  was  to  follow.  Before 
regularly  beginning  our  studies,  therefore,  it  may  be  of 
advantage  to.  acquaint  ourselves  with  the  more  prominent 
of  those  mves$igutions. 

; Antony  van  Leeuwenhoek,  the  first  to  describe  the  bodies 
now  recognized  as  bacteria,  was  born  at  Delft,  in  Holland, 
in  1632.  He  was  not  considered  a  man  of  liberal  education, 
having  been  during  his  early  years  an  apprentice  to  a  linen- 
draper.  During  his  apprenticeship  he  learned  the  art  of 
lens-grinding,  in  which  he  became  so  proficient  that  he 
eventually  perfected  a  simple  lens  by,  means  of  which  he  was 
enabled  to  see  objects  of  much  smaller  dimensions  than 
any  hitherto  seen  with  the  best  compound  microscopes  in 
existence  at  that  date.  At  the  time  of  his  discoveries  he 
was  following  the  trade  of  linendraper  in  Amsterdam. 

In  1675  he  published  the  fact  that  he  had  succeeded  in 
perfecting  a  lens  by  means  of  which  he  could  detect  in  a 
drop  of  rain-water  living,  motile  "animalcules"  of  the  most 
minute  dimensions — smaller  than  anything  that  had  hitherto 
been  seen.  Encouraged  by  this  discovery,  he  continued  to 
examine  various  substances  for  the  presence  of  what  he 
considered  animal  life  in  its  most  minute  form.  He  found 
in  sea-water,  in  well-water,  in  the  intestinal  canal  of  frogs 
and  birds,  and  in  his  own  diarrheal  evacuations,  objects 
that  differentiated  themselves  the  one  from  ,  the  other, 
not  only  by  their  shape  and  size,  but  also  by  the  peculiarity 
of  motility  which  some  of  them  were  seen  to  possess.  In  the 
year  1683  he  discovered  in  the  tartar  scraped  from  between 


INTRODUCTION  19 

the  teeth  a  form  of  microorganism  upon  which  he  laid 
special  stress.  This  observation  he  embodied  in  the  form 
of  a  contribution  to  the  Royal  Society  of  London  on  Sep- 
tember 14,  1683.  This  paper  is  of  peculiar  importance, 
not  only  because  of  the  careful,  objective  nature  of  the 
description  given  of  the  bodies  seen  by  him,  but  also  for 
the  illustrations  which  accompany  it.  From  a  perusal  of 
the  text  and  an  inspection  of  the  plates  there  remains  little 
room  for  doubt  that  Leeuwenhoek  saw  with  his  primitive 
lens  the  bodies  now  recognized  as  bacteria.1 

Upon  seeing  these  bodies  he  was  apparently  very  much 
impressed,  for  he  writes:  "With  the  greatest  astonishment 
I  observed  that  everywhere  throughout  the  material  which 
I  was  examining  were  distributed  animalcules  of  the  most 
microscopic  dimensions,  which  moved  themselves  about  in 
a  remarkably  energetic  way." 

This  discovery  was  shortly  followed  by  others  of  an 
equally  important  nature.  His  field  of  observation  appears 
to  have  increased  rapidly,  for  after  a  time  he  speaks  of  bodies 
of  much  smaller  dimensions  than  those  at  first  described  by 
him. 

Throughout  all  of  Leeuwenhoek's  work  there  is  a  con- 
spicuous absence  of  the  speculative.  His  contributions  are 
remarkable  for  their  purely  objective  nature. 

After  the  presence  of  these  organisms  in  water,  in  the 
mouth,  and  in  the  intestinal  evacuations  was  made  known 
to  the  world,  it  is  not  surprising  that  they  were  immediately 
seized  upon  as  the  explanation  of  the  origin  of  many  obscure 
diseases.  So  universal  became  the  belief  in  a  causal  relation 
between  the  "animalcules"  and  disease  that  it  amounted 

1  See  Arcana  Naturae  detecta  ab  ANTONIO  VAN  LEEUWENHOEK;  Delphis 
Batavorum,  1695. 


20  BACTERIOLOGY 

almost  to  a  germ-mania.  It  became  the  fashion  to  suspect 
the  presence  of  these  organisms  in  all  forms  and  kinds  of 
disease,  simply  because  they  had  been  demonstrated  in 
the  mouth,  intestinal  evacuations,  and  water. 

Though  nothing  of  value  at  the  time  had  been  done  in 
the  way  of  classification,  and  even  less  in  separating  and 
identifying  the  members  of  this  large  group,  still  the  fore- 
most men  of  the  day  did  not  hesitate  to  ascribe  to  them  not 
only  the  property  of  producing  pathological  conditions, 
but  some  even  went  so  far  as  to  hold  that  variations  in  the 
symptoms  of  disease  were  the  result  of  differences  in  the 
behavior  of  the  microorganisms  in  the  tissues. 

Marcus  Antonius  Plenciz,  a  physician  of  Vienna  in  1762, 
declared  himself  a  firm  believer  in  the  work  of  Leeuwenhoek, 
and  based  the  doctrine  which  he  taught  upon  the  discoveries 
of  the  Dutch  observer  and  upon  observations  of  a  confirma- 
tory nature  which  he  himself  had  made.  The  doctrine  of 
Plenciz  assumed  a  causal  relation  between  the  microorgan- 
isms discovered  and  described  by  Leeuwenhoek  and  all 
infectious  diseases.  He  maintained  that  the  material  of 
infection  could  be  nothing  else  than  a  living  substance, 
and  endeavored  on  these  grounds  to  explain  the  variations 
in  the  period  of  incubation  of  the  different  infectious  diseases. 
He  likewise  believed  the  living  contagium  to  be  capable  of 
multiplication  within  the  body,  and  spoke  of  the  possibility 
of  its  transmission  through  the  air.  He  believed  in  the 
existence  of  a  special  germ  for  each  disease,  holding  that 
just  as  from  a  given  cereal  only  one  kind  of  grain  can  grow, 
so  by  the  special  germ  for  each  disease  only  that  disease 
can  be  produced. 

He  found  in  all  decomposing  matters  innumerable  minute 
"animalculse,"  and  was  so  firmly  convinced  of  their  etio- 


INTRODUCTION  21 

logical  relation  to  the  process  that  he  formulated  the  law: 
that  decomposition  can  only  take  place  when  the  decompos- 
able material  becomes  coated  with  a  layer  of  the  organisms, 
and  can  proceed  only  when  they  increase  and  multiply. 

However  convincing  the  arguments  of  Plenciz  may  appear, 
they  seem  to  have  been  lost  sight  of  in  the  course  of  subse- 
quent events,  and  by  a  few  were  even  regarded  as  the  pro- 
ductions of  an  unbalanced  mind.  For  example,  as  late  as 
1820  we  find  Ozanam  expressing  himself  on  the  subject  as 
follows:  "Many  authors  have  written  concerning  the 
animal  nature  of  the  contagion  of  disease;  many  have 
indeed  assumed  it  to  be  developed  from  animal  substances, 
and  that  it  is  itself  animal  and  possesses  the  property  of 
life;  I  shall  not  waste  time  in  effort  to  refute  these  absurd 
hypotheses." 

Similar  expressions  of  opinion  were  heard  from  many 
other  investigators  of  the  time,  all  tending  in  the  same 
direction,  all  doubting  the  possibility  of  these  microscopic 
creatures  belonging  to  the  world  of  living  things. 

It  was  not  until  between  the  fourth  and  fifth  decades  of 
the  nineteenth  century  that  by  the  fortunate  coincidence 
of  a  number  of  important  discoveries  the  true  relation  of 
the  lower  organisms  to  infectious  diseases  was  scientifically 
pointed  out.  With  the  fundamental  investigations  of 
Pasteur  upon  the  souring  and  putrefaction  of  beer  and  wine; 
with  the  discovery  by  Pollender  and  Davaine  of  the  presence 
of  rod-shaped  organisms  in  the  blood  of  animals  dead  of 
splenic  fever,  and  with  the  progress  of  knowledge  upon  the 
parasitic  nature  of  certain  diseases  of  plants,  the  old  question 
of  "  contagium  animatum"  again  began  to  receive  attention. 
It  was  taken  up  by  Henle,  and  it  was  he  who  first  logically 
taught  this  doctrine  of  infection. 


22  BACTERIOLOGY 

The  main  point,  however,  that  had  occupied  the  attention 
of  scientific  men  from  time  to  time  for  a  period  of  about 
two  hundred  years  subsequent  to  Leeuwenhoek's  discoveries 
was  the  origin  of  the  "animalcules."  Do  they  generate 
spontaneously,  or  are  they  the  descendants  of  preexisting 
creatures  of  the  same  kind?  was  the  all-important  question. 
Among  the  earlier  participants  in  this  discussion  were  many 
of  the  most  distinguished  men  of  the  day. 

In  1749  Needham,  who  held  firmly  to  the  opinion  that 
the  bodies  which  were  attracting  such  general  attention 
developed  spontaneously  as  the  result  of  vegetative  changes 
in  the  substances  in  which  they  were  found,  attempted  to 
demonstrate  by  experiment  his  reasons  for  holding  this  view. 
He  maintained  that  the  bacteria  which  appeared  about  a 
grain  of  barley  germinating  in  a  carefully  covered  watch- 
crystal  of  water  were  the  result  of  changes  going  on  in  the 
barley-grain  itself,  incidental  to  its  germination. 

Spallanzani,  in  1769,  drew  attention  to  the  laxity  of 
Needham's  experimental  methods,  and  demonstrated  that 
if  infusions  of  decomposable  vegetable  matter  be  placed  in 
flasks,  which,  alter  being  hermetically  sealed,  were  heated 
for  a  time  in  boiling  water,  no  living  organisms  would  be 
detected  in  them,  nor  would  decomposition  appear  in  the 
infusions  so  treated.  The  objection  raised  by  Treviranus, 
viz.,  that  the  high  temperature  to  which  the  infusions  had 
been  subjected  had  so  altered  them  and  the  air  about  them 
that  the  conditions  favorable  to  spontaneous  generation 
no  longer  existed,  was  promptly  met  by  Spallanzani  when 
he  gently  tapped  one  of  the  flasks  that  had  been  boiled 
against  a  hard  object  until  a  minute  crack  was  produced; 
invariably  organisms  and  decomposition  appeared  in  the 
flask  thus  treated. 


INTRODUCTION  23 

From  the  time  of  the  experiments  of  Spallanzani  until 
as  late  as  1836  but  little  advance  was  made  in  the  elucida- 
tion of  this,  at  that  time,  obscure  problem. 

In  1836  Schulze  attracted  attention  to  the  subject  by 
the  convincing  nature  of  his  investigations.  He  showed  that 
if  the  air  which  gained  access  to  boiled  infusions  be  robbed 
of  its  living  organisms  by  first  passing  it  through  strong 
acid  or  alkaline  solutions  no  decomposition  occurred,  and 
living  organisms  could  not  be  detected  in  the  infusions. 
Following  quickly  upon  this  contribution  came  Schwann, 
in  1837,  and  somewhat  later  (1854)  Schroder  and  Dusch, 
with  similar  results  obtained  by  somewhat  different  means. 
Schwann  deprived  the  air  which  passed  to  his  infusions  of 
its  living  particles  by  conducting  it  through  highly  heated 
tubes;  whereas  Schroder  and  Dusch,  by  means  of  cotton- 
wool interposed  between  the  boiled  infusions  and  the  outside 
air,  robbed  the  air  passing  to  the  infusions  of  its  organisms 
by  the  simple  process  of  filtration.  In  1860  Hoffmann  and 
in  1861  Chevreul  and  Pasteur  demonstrated  that  the  pre- 
cautions taken  by  preceding  investigators  for  rendering 
the  air  which  entered  these  flasks  free  from  bacteria  were 
not  necessary;  that  all  that  was  required  to  prevent  the 
access  of  bacteria  to  the  infusions  in  the  flasks  was  to  draw 
out  the  neck  of  the  flask  into  a  fine  tube,  bend  it  down  along 
the  side  of  the  flask,  and  then  bend  it  up  again  a  few  cen- 
timeters from  its  extremity,  and  leave  the  mouth  open.  The 
infusion  was  then  to  be  boiled  in  the  flask  thus  prepared 
and  the  mouth  of  the  tube  left  open.  The  organisms  which 
now  fell  into  the  open  end  of  the  tube  were  arrested  by  the 
drop  of  water  of  condensation  which  collected  at  its  lowest 
angle,  and  none  could  enter  the  flask. 

While,  from  our  modern  standpoint,  the  results  of  these 


24  BACTERIOLOGY 

investigations  seem  to  be  of  a  most  convincing  nature,  yet 
there  were  many  at  the  time  who  required  additional  proof 
that  "spontaneous  generation"  was  not  the  explanation 
for  the  mysterious  appearance  of  these  minute  living  crea- 
tures. The  majority,  if  not  all,  of  such  doubts  were  sub- 
sequently dissipated  through  the  well-known  investigations 
of  Tyndall  upon  the  floating  matters  of  the  air.  In  these 
studies  he  demonstrated  by  numerous  ingenious  and  instruc- 
tive experiments  that  the  presence  of  living  organisms  in 
decomposing  fluids  was  always  to  be  explained  either  by 
the  preexistence  of  similar  living  forms  in  the  infusion 
or  upon  the  walls  of  the  vessel  containing  it,  or  by  the 
infusion  having  been  exposed  to^air  which  had  not  been 
deprived  of  its  viable  organisms. 

Throughout  all  the  work  bearing  upon  this  subject,  from 
the  time  of  Spallanzani  to  that  of  Tyndall,  certain  irregu- 
larities were  constantly  appearing.  It  was  found  that  par- 
ticular substances  required  to  be  heated  for  a  much  longer 
time  than  was  needed  to  render  other  substances  free  from 
living  organisms,  and  even  after  heating  under  the  most 
careful  precautions  decomposition  would  occasionally  occur. 

In  1762  Bonnet,  who  was  deeply  interested  in  this  sub- 
ject, suggested,  in  reference  to  the  results  obtained  by 
Needham,  the  possibility  of  the  existence  of  "germs  or 
their  eggs,"  which  had  the  power  to  resist  the  temperature 
to  which  some  of  the  infusions  employed  in  Needham's 
experiments  had  been  subjected. 

More  than  a  hundred  years  after  Bonnet  had  indulged 
in  this  pure  speculation  it  became  the  happy  privilege  of 
Ferdinand  Cohn,  of  Breslau,  to  demonstrate  its  accuracy 
and  importance. 

Cohn  repeated  the  foregoing  experiments  with  like  results. 


INTRODUCTION  25 

He  concluded  that  the  irregularities  could  only  be  due  to 
either  the  existence  of  more  resistant  species  of  bacteria 
or  to  more  resistant  stages  into  which  certain  bacteria  have 
the  property  of  passing.  He  demonstrated  that  some  of 
the  rod-shaped  organisms  possess  the  power  of  passing  into 
a  resting-  or  spore-stage  in  the  course  of  their  life-cycle, 
analogous  to  the  seeding  stage  of  higher  plants,  and  when  in 
this  stage  they  are  much  less  susceptible  to  the  deleterious 
action  of  high  temperatures  than  when  they  are  growing 
as  normal  vegetative  forms.  With  the  discovery  of  these 
more  resistant  spores  the  doctrine  of  spontaneous  generation 
received  its  death-blow.  It  was  no  longer  difficult  to  explain 
the  inconsistencies  in  the  results  of  former  investigations, 
nor  was  it  any  longer  to  be  doubted  that  putrefaction  and 
fermentation  were  the  result  of  bacterial  life  and  not  the 
cause  of  it,  and  that  these  bacteria  were  the  offspring  of 
preexisting  similar  forms.  In  other  words,  the  law  of 
Harvey,  Omne  vivum  ex  ovo,  or  its  modification,  Omne 
vivum  ex  vivo,  was  shown  to  apply  not  only  to  the  more 
highly  organized  members  of  the  animal  and  vegetable 
kingdoms,  but  to  the  most  microscopic,  unicellular  creatures 
as  well. 

The  establishment  of  this  point  gave  an  impetus  to 
further  investigations,  and  as  the  all-important  question 
was  that  concerning  the  relation  of  the  microscopic  organ- 
isms to  disease,  attention  naturally  turned  into  this  channel 
of  study.  Even  before  the  hypothesis  of  spontaneous 
generation  had  received  its  final  refutation  a  number  of 
observations  of  a  most  important  nature  had  been  made 
by  investigators  who  had  long  since  ceased  to  consider 
spontaneous  generation  as  a  tenable  explanation  of  the 
origin  of  the  microscopic  living  particles. 


26  BACTERIOLOGY 

In  the  main,  these  studies  had  been  conducted  upon 
wounds  and  the  infections  to  which  they  are  liable;  in 
fact,  the  evolution  of  our  knowledge  of  bacteriology  to  its 
present  development  is  so  intimately  associated  with  this 
particular  line  of  investigation  that  a  few  historical  facts 
in  connection  with  it  may  not  be  without  interest. 

The  observations  of  Rindfleisch,  in  1866,  in  which  he 
describes  the  presence  of  small,  pin-head  points  in  the 
myocardium  and  general  musculature  of  individuals  that 
had  died  as  a  result  of  infected  wounds,  represent,  probably, 
the  first  reliable  contribution  to  this  subject.  He  studied 
the  tissue-changes  round  about  these  points  up  to  the 
stage  of  miliary  abscess-formation.  He  refers  to  the  organ- 
isms as  "vibrios."  Almost  simultaneously  von  Reckling- 
hausen  and  Waldeyer  described  similar  changes  that  they 
had  observed  in  pyemia  and,  occasionally,  secondary  to 
typhoid  fever.  Von  Recklinghausen  believed  the  granules 
seen  in  the  abscess-points  to  be  micrococci  and  not  tissue- 
detritus,  and  gave  as  the  reason  that  they  were  regular  in 
size  and  shape,  and  gave  specific  reactions  with  particular 
straining-fluids.  Birch-Hirschfeld  was  able  to  trace  bacteria 
found  in  the  blood  and  organs  to  the  wound  as  the  point 
of  entrance,  and  believed  both  the  local  and  the  constitu- 
tional conditions  to  stand  in  direct  ratio  to  the  number  of 
spherical  bacteria  present  in  the  wound.  He  observed  also 
that  as  the  organisms  increased  in  number  they  could  often 
be  found  within  the  bodies  of  pus  corpuscles.  His  studies 
of  pyemia  led  him  to  the  important  conclusion  that  in  this 
condition  microorganisms  were  always  present  in  the 
blood. 

Of  immense  importance  to  the  subject  were  the  investiga- 
tions of  Klebs,  made  at  the  Military  Hospital  at  Karlsruhe 


INTRODUCTION  27 

in  1870-71.  He  not  only  saw,  as  others  before  him  had 
seen,  that  bacteria  were  present  in  diseases  following  infec- 
tion of  wounds,  but  described  the  manner  in  which  the 
organisms  had  gained  entrance  from  the  point  of  injury 
to  the  internal  organs  and  blood.  He  expressed  the  opinion 
that  the  spherical  and  rod-shaped  bodies  which  he  saw  in 
the  secretions  of  wounds  were  closely  allied,  and  he  gave  to 
them  the  designation  "  microsporon  septicum."  He  believed 
that  the  organisms  gained  access  to  the  tissues  round  about 
the  point  of  injury  both  by  the  aid  of  the  wandering  leuko- 
cytes and  by  being  forced  through  the  connective-tissue 
lymph-spaces  by  the  mechanical  pressure  of  muscular 
contraction. 

On  erysipelatous  inflammations  secondary  to  injury 
important  investigations  were  also  being  made,  Wilde, 
Orth,  von  Recklinghausen,  Lukomsky,  Billroth,  Ehrlich, 
Fehleisen,  and  others  agreeing  that  in  these  conditions 
microorganisms  could  always  be  detected  in  the  lymph 
channels  of  the  subcutaneous  tissues;  and  through  the 
work  of  Oertel,  Nassiloff,  Classen,  Letzerich,  Klebs,  and 
Eberth  the  constant  presence  of  bacteria  in  the  diphtheritic 
deposits  at  times  seen  on  open  wounds  was  established. 

.  We  see  that  the  conception  of  a  living,  invisible  some- 
thing— a  contagium  vivum — was  old,  but  by  the  use  of  the 
rapidly  improving  compound  microscope  a  host  of  investi- 
gators was  making  this  "something"  more  tangible;  they 
were  describing  various  minute  bodies  seen  in  diseased 
conditions  that  they  believed  to  be  living  things,  and  to  be 
the  cause  of  the  conditions  in  which  they  were  observed. 
Yet  no  convincing  demonstration  of  the  relationship  between 
these  supposed  living  foreign  bodies  and  the  diseases  in  which 
they  were  present  had  been  made.  In  1855  Pollender 


28  BACTERIOLOGY 

announced  the  constant  presence  in  the  blood  of  animals 
dead  of  anthrax  of  rod-shaped  bodies,  and  in  1863  Davaine 
showed  the  blood  of  such  animals  to  be  infective  for  normal 
animals.  This  distinct  step  in  advance  so  attracted  the 
attention  of  Pasteur  that  he  soon  became  closely  identified 
not  only  with  studies  of  this  particular  disease  but  with 
other  diseases  of  domestic  animals  and  fowls.  Pasteur  was 
already  known  in  the  fields  of  physical  and  natural  sciences 
through  his  basic  work  on  the  organic  isomers  and  his  dis- 
coveries in  the  field  of  fermentation.  His  conception  of 
fermentation  as  a  function  of  living  cells  was  so  opposed  to 
the  views  generally  held  at  the  time,  and  especially  those 
held  by  von  Liebig — perhaps  the  most  distinguished  chem- 
ist of  his  day— that  endless  discussion,  amounting  at  times 
almost  to  polemic,  took  place.  Pasteur's  opinions  finally 
triumphed.  His  manifold  investigations  at  this  time  were 
so  novel,  so  progressive;  his  interests  so  versatile  and  so  in 
accord  with  the  new  thought  that  was  beginning  to  develop, 
that  he  was  soon  regarded  as  the  most  suggestive  and  impres- 
sive contributor  of  his  time.  His  subsequent  studies  upon 
vaccination  with  living  attenuated  viruses  were  of  such 
fundamental  nature  both  scientifically  and  practically  as 
to  justify  the  opinion  that  the  science  of  immunology  began 
with  his  observations  and  investigations.  The  circumstances 
surrounding  his  public  demonstration  that  sheep  can  be 
protected  from  anthrax  infection  by  the  process  of  vaccina- 
tion with  a  living,  attenuated  virus  constitutes  one  of  the 
most  dramatic  incidents  in  the  history  of  applied  science. 

The  catholicity  of  Pasteur's  interests  in  all  matters  con- 
cerning the  world  of  living,  microscopic  things,  the  benefits 
that  accrued  from  the  application  of  his  keen  analytic  men- 
tality to  the  solution  of  problems  of  domestic,  agricultural 


LOUIS  PASTEUR 
1822-1893 


INTRODUCTION  29 

and  commercial  moment,  and  his  contributions  to  our 
understanding  of  infection,  transmission  and  induced 
immunity  from  disease,  justify  the  statement  that  he  was 
easily  our  broadest  minded  and  most  comprehensive  con- 
tributor to  the  field  of  microbiology.1 

Simple  and  natural  as  all  this  may  seem  to  us  now,  the 
stage  to  which  the  subject  had  developed  when  these  obser- 
vations were  recorded  did  not  admit  of  their  meeting  with 
unconditional  acceptance.  The  only  strong  argument  in 
favor  of  the  etiological  relation  of  the  organisms  that  had 
been  seen  to  the  diseases  with  which  they  were  associated 
was  the  constancy  of  this  association  and  the  occasional 
transmission  of  the  disease  from  a  sick  to  a  well  animal  by 
the  use  of  body  fluids  or  bits  of  diseased  tissue.  No  efforts 
had  been  made  to  isolate  them,  and  but  few  to  reproduce  the 
pathological  conditions  by  inoculation.  Moreover,  not  a 
small  number  of  investigators  were  skeptical  as  to  the 
importance  of  such  demonstrations;  many  claimed  that 
microorganisms  were  normally  present  in  the  blood  and 
tissues  of  the  body;  and  some  even  urged  that  the  organisms 
seen  in  diseased  conditions  were  the  result  rather  than  the 
cause  of  the  maladies.  It  is  hardly  necessary  to  do  more 
than  say  that  both  of  these  views  were  purely  speculative, 
and  have  never  had  a  single  reliable  experimental  argument 
in  their  favor.  Billroth  and  Tiegel,  who  held  to  the  former 
opinion,  did  endeavor  to  prove  their  position  through  experi- 
mental means;  but  the  methods  employed  by  them  were  of 
such  an  untrustworthy  nature  that  the  fallacy  of  deductions 
drawn  from  them  was  very  quickly  made  manifest  by  subse- 
quent investigators.  Their  method  for  demonstrating  the 

1  See  Life  of  Pasteur,  by  Vallery-Radot. 


30  BACTERIOLOGY 

presence  of  microorganisms  in  normal  tissues  was  to  remove 
bits  of  organs  from  the  healthy  animal  body  with  heated 
instruments  and  drop  them  into  hot  melted  paraffin.  They 
held  that  all  living  organisms  on  the  surface  of  the  tissues 
would  be  destroyed  by  the  high  temperature,  and  that  if 
decomposition  should  subsequently  occur  it  would  prove  that 
it  was  the  result  of  the  growth  of  bacteria  in  the  depths  of 
the  tissues  to  which  the  heat  had  not  penetrated.  Decom- 
position did  usually  set  in,  and  they  accepted  this  as  proof  of 
the  accuracy  of  their  view.  Attention  was,  however,  shortly 
called  to  the  fact  that  in  cooling  there  was  contraction  of 
paraffin,  resulting  usually  in  the  production  of  small  rents 
and  cracks  in  which  dust,  and  bacteria  lodged  upon  it, 
could  accumulate  and  finally  gain  access  to  the  tissues, 
with  the  occurrence  of  decomposition  as  a  consequence. 
Their  results  were  thus  explained  after  a  manner  analogous 
to  that  employed  by  Spallanzani,  in  1769,  in  demonstrating 
to  Treviranus  the  fallacy  of  the  opinion  held  by  him  and  the 
accuracy  of  his  own  views,  viz.,  that  it  was  always  through 
the  access  of  organisms  from  without  that  decomposition 
primarily  originated.  (See  page  22.) 

Under  careful  precautions,  to  which  no  objection  could 
be  raised,  the  experiments  of  Billroth  and  Tiegel  w^re 
repeated  by  Pasteur,  Burdon-Sanderson,  and  Klebs,  but 
with  failure  in  every  instance  to  demonstrate  the  presence 
of  bacteria  in  the  healthy  living  tissues. 

The  fundamental  researches  of  Koch  (1881)  upon  patho- 
genic bacteria  and  their  relation  to  the  infectious  diseases 
of  animals  differed  from  those  of  preceding  investigators 
in  many  important  respects.  The  scientific  methods  of 
analysis  with  which  each  and  every  obscure  problem  was 
met  as  it  arose  served  at  once  to  distinguish  him  as  a  pioneer 


ROBERT  KOCH 
1843-191O 


INTRODUCTION  31 

in  this  hitherto  but  imperfectly  cultivated  domain.  The 
outcome  of  these  investigations  was  the  establishment  of 
a  foundation  upon  which  practical  bacteriology  of  the  future 
was  to  rest.  He,  for  the  first  time,  demonstrated  that  dis- 
tinct varieties  of  infection,  as  evidenced  by  anatomical 
changes  are  due  in  many  cases  to  the  activities  of  specific 
microorganisms,  and  that  by  proper  methods  it  is  possible 
to  isolate  these  organisms  in  pure  culture,  to  cultivate  them 
indefinitely  under  artificial  conditions,  to  reproduce  the 
lesions  by  inoculation  of  these  pure  cultures  into  susceptible 
animals,  and  to  continue  the  disease  at  will  by  continuous 
inoculation  from  an  infected  to  a  healthy  animal. 

By  the  methods  that  he  employed  he  demonstrated  a 
series  of  separate  and  distinct  diseases  that  can  be  produced 
in  mice  and  rabbits  by  the  injection  of  putrid  substances 
into  their  tissues.  The  disease  known  as  septicemia  of 
mice;  likewise  a  disease  characterized  by  progressive  abscess 
formation,  and  pyemia  and  septicemia  of  rabbits,  were  among 
the  affections  first  produced  by  him  in  this  way.  It  was  in 
the  course  of  this  work  that  the  Abbe  system  of  substange 
condensing  apparatus  was  first  used  in  bacteriology;  that 
the  aniline  dyes  suggested  by  Weigert  were  brought  into 
general  use;  that  the  isolation  and  cultivation  of  bacteria  in 
pure  culture  on  solid  media  were  shown  to  be  possible;  and 
that  animals  were  employed  as  a  means  of  obtaining  from 
mixtures  pure  cultures  of  pathogenic  bacteria. 

With  the  bounteous  harvest  of  original  and  important 
suggestions  that  was  reaped  from  Koch's  classical  series 
of  investigations  bacteriology  reached  an  epoch  in  its  devel- 
opment, and  at  this  period  practical  bacteriology,  as  we 
know  it  today,  may  justly  be  said  to  have  had  its  birth. 


32  BACTERIOLOGY- 

NOTE. — I  have  presented  only  the  most  prominent  inves- 
tigations that  will  serve  to  indicate  the  lines  along  which 
the  subject  has  developed.  For  a  more  detailed  account 
of  the  historical  development  of  the  work  the  reader  is 
referred  to  Loffler's  instructive  and  entertaining  Vorlesungen 
uber  die  geschichtliche  Entwickelung  der  Lehre  von  den  Bac- 
terien,  upon  which  I  have  drawn  freely  in  preparing  the 
foregoing  sketch. 


CHAPTER  I. 

Definition  of  Bacteria — Differences  Between  Parasites  and  Saprophytes 
— Their  Place  in  Nature — Bacterial  Enzymes — Products  of  Bacteria 
— Nutrition  of  Bacteria — Their  Relation  to  Oxygen — Influence  of 
Temperature  Upon  Their  Growth — Chemotaxis. 

BACTEKIA  (more  properly  bacteriacese  or  schizomycetes) 
were  regarded  by  the  older  writers  as  infusoria.  This  was 
because  of  their  capacity  for  developing  in  infusions,  their 
property  of  spore  formation,  their  resistance  to  drying, 
their  power  of  independent  motion,  and  the  absence  of 
chlorophyl  from  their  tissues.  In  the  modern  conception, 
however,  this  classification  is  untenable,  and  bacteria,  by 
virtue  of  their  distinguishing  peculiarities,  are  now  treated 
as  a  group  by  themselves  that  may  briefly  be  defined  as 
comprising  microscopic,  unicellular,  vegetable  organisms 
that  multiply  by  the  process  of  transverse  division. 

Inasmuch  as  bacteria  are  not  possessed  of  chlorophyl, 
their  metabolic  processes  are  fundamentally  different  from 
those  of  the  higher  plants  in  which  it  is  present.  They 
cannot,  as  in  the  case  of  the  green  plants,  obtain  carbon 
and  nitrogen  from  such  simple  bodies  as  carbon  dioxide  and 
ammonia,  but  are  forced  to  secure  these  essential  elements 
from  organic  matter  as  such.  This  power  to  decompose  and 
assimilate  organic  matters  is  signally  different  in  different 
species  of  bacteria,  and,  singular  to  say,  there  is  a  small 
group  (to  be  described  later)  from  which  this  function  is 
apparently  absent,  in  spite  of  the  fact  that  no  compensatory 
chlorophyl  is  discernible  in  their  tissues. 

3  (33) 


34  BACTERIOLOGY 

SAPROPHYTES  AND  PARASITES.  —  In  the  case  of  certain 
bacteria,  in  fact,  the  majority,  the  source  of  food  supply 
must  of  necessity  be  dead  organic  matters  of  either  animal 
or  vegetable  origin.  They  cannot  exist  in  the  presence  of 
living  tissues.  To  the  members  of  this  group  the  designa- 
tion saprophytic  or  metatrophic  (A.  Fischer)  is  given.  To 
that  group  that  can  exist  only  upon  living  organic  matters, 
and  herein  belong  many  (not  all)  of  the  disease-producing 
bacteria,  the  appellation  parasitic  or  paratrophic  (A.  Fischer) 
is  applied;  while  for  the  few  species  that  either  do  not 
require  organic  matters,  or  do  not,  so  far  as  is  known,  have 
the  faculty  of  decomposing  and  assimilating  proteid  stuffs 
at  all,  the  name  prototrophic  is  suggested  by  Fischer.  In 
the  strict  sense  of  the  word,  a  parasite  can  exist  only  in  the 
body  of  a  living  host,  and  a  saprophyte  only  upon  lifeless 
organic  matters,  and  such  obligate  parasites  and  saphrophytes 
are  known,  but  in  the  majority  of  cases  such  nutritive  con- 
ditions are  not  obligatory,  many  of  both  parasites  and 
saprophytes  having  the  power  to  adapt  themselves  to 
conditions  other  than  those  for  which  they  are  by  nature 
best  fitted.  For  instance,  certain  species  that  exhibit  their 
most  important  properties  under  conditions  of  parasitism 
may,  nevertheless,  lead  a  saprophytic  existence  when  cir- 
cumstances demand  it,  and,  on  the  other  hand,  particular 
species  usually  saprophytic  by  nature  may  find  conditions 
favorable  to  their  development  in  a  living  host.  To  such 
adaptable  species  the  designation  "facultative"  is  given, 
and,  when  employed,  signifies  that  the  species  in  question 
has  the  faculty  of  adapting  itself  to  environments  other 
than  those  in  which  it  is  usually  encountered.  In  this  sense 
all  of  the  disease-producing  bacteria  that  can  be  cultivated 
artificially  are  manifestly  facultative  saprophytes. 


DEFINITION  OF  BACTERIA  35 

The  life-processes  of  bacteria  are  so  rapid,  complex,  and 
energetic  that  they  result  in  the  most  profound  alterations 
in  the  structure  and  composition  of  the  materials  in  and 
upon  which  they  are  developing. 

Disintegrations  and  decompositions  result  from  the  acti- 
vities of  the  saprophytic  bacteria;  while  the  changes  brought 
about  in  the  tissues  of  their  living  host  by  the  purely  parasitic 
forms  find  expression  in  disease-processes  not  infrequently 
leading  to  complete  death. 

THEIR  PLACE  IN  NATURE. — The  role  played  in  nature  by 
the  saprophytes  is  a  very  important  one.  Through  their 
functional  activities  the  highly  complicated  tissues  of  dead 
animals  and  vegetables  are  resolved  into  the  simpler  com- 
pounds, carbonic  acid,  water,  and  ammonia,  in  which  form 
they  may  be  taken  up  and  appropriated  as  nourishment  by 
the  more  highly  organized  members  of  the  vegetable  king- 
dom. It  is  through  this  ultimate  production  of  carbonic 
acid,  ammonia,  and  water  by  bacteria,  as  end-products  in 
the  processes  of  decomposition  and  fermentation  of  dead 
animal  and  vegetable  tissues,  that  the  demands  of  growing 
vegetation  for  these  compounds  are  supplied. 

The  more  highly  organized  chlorophyl  plants  do  not 
possess  the  power  of  obtaining  their  carbon  and  nitrogen 
from  such  complicated  organic  substances  as  serve  for  the 
nutrition  of  bacteria,  and  as  the  production  of  the  simpler 
compounds,  carbon  dioxide  and  ammonia,  by  the  animal 
world  is  not  sufficient  to  meet  the  demands  of  the  chloro- 
phyl plants,  the  importance  of  the  part  played  by  bacteria 
in  making  up  this  deficit  is  obvious  and  cannot  be  overesti- 
mated. Were  it  not  for  the  activity  of  these  microscopic 
living  creatures  all  life  upon  the  surface  of  the  earth  would 
cease.  Deprive  higher  vegetation  of  the  carbon  and  nitrogen 


36  BACTERIOLOGY 

supplied  to  it  as  a  result  of  bacterial  activity,  and  its  develop- 
ment comes  rapidly  to  an  end;  rob  the  animal  kingdom  of 
the  food-stuffs  supplied  to  it  by  the  vegetable  world,  and 
life  is  no  longer  possible.  It  is  plain,  therefore,  that  in  this 
cycle  of  life  phenomenon  the  saprophytes,  which  represent 
the  large  majority  of  all  bacteria,  must  be  looked  upon  in 
the  light  of  benefactors,  without  which  existence  would  be 
impossible. 

With  the  parasites,  on  the  other  hand,  the  conditions  are 
far  from  analagous.  Through  their  metabolic  activities 
there  is  constantly  a  loss,  rather  than  a  gain,  to  both  the 
animal  and  vegetable  kingdoms.  Their  host  must  always 
be  a  living  body  in  which  exist  conditions  favorable  to  their 
development,  and  from  which  they  appropriate  substances 
that  are  necessary  to  the  health  and  life  of  the  organism  on 
which  they  are  preying;  at  the  same  time  they  elaborate 
substances  as  products  of  their  nutrition  that  are  directly 
poisonous  to  the  tissues  in  which  they  are  growing. 

In  their  relations  to  terrestrial  life,  therefore,  the  posi- 
tions occupied  by  the  two  functionally  different  groups,  the 
saprophytes  on  the  one  hand,  and  the  parasites  on  the 
other,  are  diametrically  opposed. 


SPECIFIC  FUNCTIONS  OF  SAPROPHYTIC  BACTERIA. 

Appropriate  investigation  of  the  saprophytic  group  of 
bacteria  has  shed  important  light  upon  certain  specific 
characteristics  with  which  many  of  the  species  are  endowed. 
We  know  that  numerous  common  phenomena  are  the  results 
of  their  activities.  The  souring  of  milk,  the  ripening  of 
cheese;  certain  of  the  fermentations  resulting  in  the  forma- 
tion of  various  acids  of  the  fatty  series;  the  elaboration  of 


FUNCTIONS  OF  SAPROPHYTIC  BACTERIA          37 

other  aromatic  bodies  of  organic  character  and  origin;  the 
spoiling  of  wine;  the  disintegrations  incidental  to  the 
manufacture  of  hemp  products;  the  old  method  of  making 
indigo;  the  natural  and  artificial  methods  for  the  destruction 
of  the  organic  waste  encountered  in  polluted  waters  and 
sewage  and  the  transformations  of  dead  organic  matter  in 
the  soil  are  all  illustrations  of  these  well-known  phenomena. 
In  a  number  of  commercial  lines  constant  use  is  made  of 
these  bacterial  activities.  This  is  conspicuously  seen  in 
the  manufacture  of  butter  and  cheese  where  the  excellence 
of  the  products  is  due  to  the  peculiar  flavors  caused  by 
bacterial  growth  in  the  raw  materials.  Before  synthetic 
methods  became  so  generally  in  use  bacterial  activities  were 
largely  employed  in  the  manufacture  of  the  organic  acids. 

In  addition  to  the  foregoing  a  number  of  saprophytes 
have  the  specific  property  of  producing  beautiful  pigments, 
red,  yellow,  orange,  pink,  violet,  green,  etc.  This  group  of 
"  chromogens"  as  they  are  called  have  doubtless  other  func- 
tions in  the  great  laboratory  of  nature,  the  soil,  where  they 
are  commonly  found,  but  color  production  is  the  most 
obvious. 

Another  group — the  "photogens"  or  photogenic  species 
have  the  remarkable  ability  to  produce  luminosity  in  the 
substances  in  or  on  which  they  exist.  It  is  to  the  activity 
of  this  group  that  the  phosphorescence  sometimes  seen  in 
decayed  wood,  in  rotten  fish  and  other  flesh  is  attributable. 
How  it  is  done  is  a  mystery,  just  as  is  the  means  by  which 
the  fire-fly  an,d  the  glow-worm  emit  their  tiny  sparks  of  light. 

Another  group  have  as  the  end  products  of  their  activities 
those  evil  smelling  bodies  by  which  putrefaction  is  charac- 
terized, these  are  the  so-called  "saprogenic"  species. 

Others  have  as  their  most  interesting  functions  the  power 


38  BACTERIOLOGY 

to  carry  hydrogen  sulphide  to  higher  sulphur  compounds, 
the  so-called  "thiogenic"  species. 

Those  saprophytes  that  are  concerned  in  such  well-known 
fermentations  as  result  in  the  production  of  the  various 
acids  of  the  fatty  series  are  known  as  "zymogens." 

But  of  all  the  so-called  saprophytic  group  none  are  more 
interesting  and  none  by  any  means  so  important  as  those 
concerned  in  the  various  transformations  through  which 
nitrogen  passes  in  being  prepared  as  food  for  higher  vege- 
tation. This  group,  or  rather  those  groups,  for  there  are 
apparently  several  operating  on  nitrogen  and  its  compounds 
in  various  ways,  are  known  as  the  "nitrifying"  and  the 
"denitrifying"  and  the  " nitrogen  fixing"  bacteria. 

NITRIFYING  BACTERIA. — They  carry  ammonia,  resulting 
from  the  decomposition  of  dead  animals  and  plants;  by 
a  process  of  oxidation  first  to  nitrous  acid,  then  by  further 
oxidation  the  nitrous  acid  is  carried  to  nitric  acid.  These 
two  steps  in  the  process  are  taken  by  two  totally  distinct 
groups  of  bacteria  of  a  most  interesting  nature.  The  func- 
tion of  one  group  is  strictly  limited  to  the  nitrite  process; 
that  of  the  other  to  the  nitrate;  the  latter  taking  up  the 
work  at  the  point  where  the  former  leaves  it.  The  former 
cannot  carry  its  operation  beyond  the  nitrite  point,  nor  can 
the  latter  begin  with  ammonia  and  carry  it  to  complete 
nitrifaction.  A  most  singular  peculiarity  of  this  group  is 
the  inability  to  develop  on  the  nutrient  media  commonly 
used  for  the  cultivation  of  bacteria.  Organic  matter  as 
such  seems  to  be  unfavorable  to  their  viability.  To  grow 
them  one  is  obliged  to  use  a  silicate  jelly,  a  sort  of  water 
glass  of  about  the  consistency  of  ordinary  gelatin,  to  which 
are  added  certain  salts  that  these  particular  species  are 
able  to  decompose  in  order  to  secure  the  elements  necessary 


FUNCTIONS  OF  SAPROPHYTIC  BACTERIA          39 

to  provide  energy.  In  so  far  as  life  upon  the  earth's  surface 
is  concerned  the  nitrification  going  on  in  the  soil  as  a  result 
of  the  activities  of  this  group  is  one  of  the  most  important 
phenomena  in  operation.  It  is  to  a  large  extent  responsible 
for  supplying  higher  vegetation  with  nitrogen  in  a  form 
available  for  food. 

Denitrifieation,  i.  e.,  the  reverse  of  nitrif action,  the  reduc- 
tion of  nitrates  and  nitrites  to  ammonia  is  a  function  peculiar 
to  many  bacteria,  particularly  many  species  found  in  the 
soil.  Often  it  does  not  appear  to  be  a  specific  function  and 
is  frequently  accomplished  under  conditions  where  organic 
matter  is  present  and  is  utilized.  In  many  cases  the  denitrifi- 
cation  seems  to  be  less  a  phenomenon  due  to  the  specific 
activities  of  the  bacteria  themselves  than  to  the  reducing 
action  of  the  products  of  their  growth.  In  the  case  of  the 
few  species  that  have  been  called  "true  denitrifiers,"  the 
reduction  appears  to  be  due  to  the  respiratory  demands  of 
those  species  for  oxygen;  this  robbing  of  the  oxides  of 
nitrogen  of  their  oxygen  by  the  bacteria  resulting,  mani- 
festly, in  reduction. 

Nitrogen  Fixation. — Another  phenomenon  having  to  do 
with  nitrogen,  and  resulting  from  the  activity  of  saprophytes, 
is  the  so-called  "nitrogen  fixation"  by  bacteria.  For  many 
years  we  were  taught  that  the  nitrogen  of  the  air,  constitut- 
ing about  80  per  cent,  of  the  entire  atmosphere,  was  of  no 
biological  significance  and  was  put  there  by  nature  merely  to 
dilute  the  excessively  active  oxygen  to  a  point  compatible 
with  respiration  by  man  and  animals.  This  extraordinary 
conception  was  always  looked  upon  with  suspicion  by  thought- 
ful students.  It  was  not,  however,  until  about  1886  that 
the  real  significance  of  atmospheric  nitrogen  was  made 
clear.  Hellrigel  and  Wilfarth  at  that  time  demonstrated 


40  BACTERIOLOGY 

that  the  nodules  found  on  the  roots  of  the  leguminous 
plants  (clover,  peas,  beans,  etc.),  might  properly  be  re- 
garded as  communities  of  bacteria  which  were  beneficently 
cooperating  with  the  plants  in  the  performance  of  their 
fundamental  life  processes,  i.  e.,  they  were  in  "symbiotic" 
relationship.  The  result  of  this  cooperation  they  showed 
to  be  the  power  of  the  legumens  to  fix  and  store  the  free 
atmospheric  nitrogen.  When  one  realizes  how  inexhaustible 
is  the  supply  of  free  atmospheric  nitrogen  it  is  difficult 
to  exaggerate  the  importance  of  this  function.  It  also  sheds 
interesting  light  upon  certain  practices  of  the  agriculturalist 
that  have  been  in  empirical  operation  since  the  cultivation 
of  the  soil  began.  It  has  always  been  known  that  the 
rotation  of  crops  is  essential  to  the  successful  tillage  of  the 
soil  and  we  find  that  in  such  rotation  one  or  another  of  the 
legumens  was  always  used.  The  reason  is  evident,  they  do 
not  impoverish,  but  though  their  ability  to  fix  nitrogen 
through  the  aid  of  the  "nodule  bacteria"  on  their  roots, 
they  actually  enrich  the  soil. 

From  the  foregoing  it  is  obvious  that  the  expression 
"nature's  laboratory"  is  properly  applied  to  the  soil.  It 
is  here  that  all  the  saprophytes  are  sooner  or  later  found. 
Among  her  various  analytic  and  synthetic  performances 
nature  concerns  herself  with  none  so  important  to  life  as 
those  having  to  do  with  the  several  transformations  of  nitro- 
gen to  which  allusion  has  just  been  made. 

SPECIFIC  FUNCTIONS  OF  THE  PARASITIC  BACTERIA. 

As  already  intimated  the  parasitic  bacteria  are  not  charac^ 
terized  by  such  beneficent  activities  as  are  possessed  by 
the  saprophytic  group;  they  exist  at  the  expense  of  living 


FUNCTIONS  OF  THE  PARASITIC  BACTERIA       41 

hosts  and  usually  excite  detrimental  changes  in  those  hosts. 
It  is  to  the  parasitic  group  that  the  pathogenic  or  disease- 
exciting  bacteria  belong. 

Strictly  speaking  none  of  the  pathogenic  bacteria  with 
which  we  are  acquainted  are  obligate  parasites,  that  is, 
none  of  them  grow  and  multiply  only  in  the  body  of  a  living 
host;  for  all  have  been  cultivated  under  artificial  conditions 
on  dead,  nutrient  cultural  materials.  They  are  nevertheless 
properly  classified  as  parasites  for  it  is  only  under  conditions 
of  parasitism  that  they  exhibit  those  activities  that  make 
them  the  objects  of  special  interest. 

When  circumstances  admit  of  the  various  members  of 
this  group  getting  access  to  the  living  hosts  in  which  they 
find  conditions  favorable  to  their  growth  and  multiplica- 
tion there  results  the  state  known  as  "disease."  In  some 
cases  the  disease  is  local,  i.  e.,  it  involves  only  the  tissues  in 
the  immediate  vicinity  of  the  invading  bacteria;  in  others 
it  is  general;  involves  the  entire  body  and  eventuates  in 
the  death  of  the  host. 

As  we  study  the  peculiarities  of  the  disease-producing 
bacteria  more  closely  we  find  that  in  inducing  disease  they 
do  not  all  operate  in  the  same  way,  though  the  ultimate 
forces  used  by  them  in  the  destruction  of  living  tissue  are 
throughout  analogous,  i.  e.,  they  are  poisons. 

In  some  cases  the  parasite  finds  the  circulating  fluids  of 
the  host  the  most  favorable  place  for  its  growth  and  develop- 
ment. Under  such  circumstances  it  is  not  uncommon  for 
the  blood-  and  lymphvessels  of  an  infected  animal  to  be 
almost  filled  with  the  parasites  within  a  short  tune  after 
the  invasion.  To  such  a  state  the  designation  "septicemia" 
is  given,  that  is,  there  is  a  septic  condition  of  the  blood, 
"blood  poisoning"  as  it  is  commonly  called. 


42  BACTERIOLOGY 

In  other  instances  a  parasitic  species  may  manifest  its 
activities  in  a  very  insignificant  way,  insofar  as  the  welfare 
of  the  host  is  concerned.  The  causation  of  simple  boils, 
pimples  and  unimportant  local  inflammations  serves  to 
illustrate  this.  In  other  examples  we  find  the  activities 
of  the  parasite  more  or  less  confined  to  special  vital  organs 
of  the  body,  the  restriction,  practically  speaking,  of  the 
cholera  and  dysentery  germs  to  the  mucosa  of  the  intestinal 
canal;  of  the  gonorrheal  germ  to  the  mucous  surfaces  of 
the  genito-urinary  tract;  of  the  typhoid  bacillus  to  the 
lymphatic  structures  of  the  abdominal  cavity  may  serve 
as  illustrations. 

Again  we  know  of  parasitic  species  that  do  not  disseminate 
beyond  their  portal  of  entry.  They  grow  at  that  point  and 
manufacture  deadly  poisons  which  are  disseminated  through- 
out the  body  by  way  of  the  circulating  fluids.  The  germs 
of  diphtheria  and  of  tetanus  are  striking  illustrations  of 
this  type  of  parasite. 

In  practically  all  cases  fever  is  an  accompaniment  of  the 
activities  of  the  parasitic  bacteria  in  the  body  though  in 
certain  particular  instances  an  initial  rise  in  temperature 
may  be  followed  by  marked  depressions  of  it,  due  to  the 
action  of  the  poisons  elaborated  by  the  bacteria. 

In  considering  the  activities  of  the  parasitic  group  of 
bacteria  we  encounter  at  the  beginning  one  factor  in  par- 
ticular with  which  we  are  not  called  upon  to  reckon  in  speak- 
ing of  the  saprophytic  group.  The  saprophytes  work  upon 
inert,  dead  matter;  the  parasites  on  active,  living  matter, 
all  of  which  is  by  nature  endowed  with  some  degree  of 
resistance  to  the  inroads  and  activities  of  invading  para- 
sites. It  is  through  this  "vital  resistance"  possessed  by  the 
living  host  that  many  of  the  irregularities  seen  in  the  opera- 


FUNCTIONS  OF  THE  PARASITIC  BACTERIA        43 

tion  of  the  parasitic  group  may  be  explained.  It  is  indeed 
so  active  in  certain  individual  cases  as  to  give  to  its  possessor 
almost  complete  immunity  from  particular  types  of  parasitic 
invasion. 

To  illustrate:  Diphtheria  is  caused  by  a  well-known 
parasite.  It  has  many  interesting  properties  but  the  most 
significant  of  its  physiological  activities  is  its  power  to 
elaborate  a  poison  that  causes  the  group  of  symptoms  and 
tissue  changes  which  we  know  as  diphtheria.  No  other 
organism  has  this  property.  This  same  diphtheria  bacillus 
may  invade  one  person  and  cause  his  death,  while  in  another 
the  results  of  its  activities  may  be  comparatively  trifling. 
Similar  extremes  of  variation  are  constantly  seen  in  the  case 
of  all  the  known  infective  diseases.  It  is  to  be  explained  in 
but  one  way  "the  soil,"  i.  e.,  the  living  host,  in  which  the 
disease  exciting  "  seed,"  the  germ,  finds  itself  is  not  that 
which  is  best  suited  to  its  active  growth,  or  in  other  words 
one  individual  possess  higher  natural  powers  of  resistance 
than  does  another,  and  in  a  large  group  of  individuals  such 
differences  in  the  degree  of  resistance  are  marked.  We  see 
nothing  like  this  in  the  action  of  saprophytes  upon  dead 
matter.  It  is  true  we  see  their  growth  restrained  at  times. 
In  some  cases  such  restraint  is  exercised  by  other  species 
the  products  of  whose  growth  are  antagonistic;  and  in  a 
number  of  cases  the  growth  of  saprophytes  is  often  for  a 
time  checked  by  the  accumulated  products  of  their  own 
activities.  For  instance  the  growth  of  those  saprophytes 
concerned  in  acid  fermentations  comes  very  quickly  to  an 
end  unless  special  provisions  be  made  to  neutralize  and  fix 
the  acids  as  fast  as  they  are  manufactured,  for  no  bacteria 
develop  indefinitely  in  the  presence  of  free  acids.  This  is, 
however,  a  very  different  kind  of  inhibition  from  that 


44  BACTERIOLOGY 

exercised   by   living   tissues   in   repelling   the   invasion   of 
parasites. 

In  the  foregoing  brief  sketch  of  the  manifold  transfor- 
mation resulting  from  bacterial  activity  there  is  no  dis- 
cussion of  the  mechanism  through  which  such  changes  are 
wrought.  In  the  case  of  the  saprophytes  the  various  analyses 
and  syntheses  that  accompany  their  growth  are  in  general 
believed  to  be  manifestations  of  fermentations;  while  the 
activities  of  the  parasites  in  producing  disease  are  referred 
to  poisons  elaborated  by  these  that  have  a  destructive  action 
upon  the  tissues  in  which  the  parasites  are  operating.  In 
the  following  paragraphs  an  effort  will  be  made  to  elucidate 
this  phase  of  the  subject. 

FERMENTS,  ENZYMES,  TOXINS,  BACTERIAL  PROTEINS, 
AND  PTOMAINS. 

There  is  perhaps  no  department  of  either  biology  or  physics 
that  relates  to  more  important  phenomena,  more  widespread 
phenomena  or  more  inexplicable  phenomena  than  that 
having  to  do  with  fermentation  and  the  agencies  that  cause 
it. 

The  phenomenon  has  attracted  the  attention  of  the  ablest 
investigators  for  years  and  we  are  scarcely  nearer  to  an 
understanding  of  its  intimate  nature  today  than  we  were 
at  the  beginning. 

In  its  older  sense,  the  word  fermentation  related  to  all 
reactions  that  are  accompanied  by  the  evolution  of  gas  and, 
indeed,  it  is  probable  that  the  word  originated  with  the  word 
fervere,  meaning  to  seethe,  to  boil,  to  bubble.  In  its  modern 
usage,  however,  the  word  comprehends  many  reactions, 
believed  to  be  caused  by  ferments,  but  during  which  no 


FERMENTS,  ENZYMES,   TOXINS  AND  PTOMAINS     45 

gas  as  such  is  evolved:  The  fermentation  best  and  longest 
known  to  man  is  that  through  which  sugar  is  converted 
into  alcohol,  seen  in  the  making  of  wine  from  grapes.  In 
so  far  as  bacteria  are  concerned  we  are  aware  of  a  multipli- 
city of  reactions  which  are  believed  to  be  manifestations 
of  fermentation,  though  opinion  on  these  points  is  far  from 
being  in  agreement.  However,  as  ferments  have  never 
been  isolated  in  a  pure  state  and  as  the  real  nature  of  their 
activities  cannot  with  the  present  means  at  our  disposal, 
be  finally  determined,  there  is  as  much  justification  for 
regarding  such  reactions  as  excited  by  ferments  as  not. 
We  shall  therefore  assume  that  both  the  normal  metabolism 
of  the  bacterial  cell  and  its  peculiar  power  to  excite  specific 
reactions  in  various  substances  are  made  possible  through 
the  agency  of  ferments.  In  some  cases  such  ferments  are 
firmly  bound  up  as  integral  parts  of  the  cell  protoplasm. 
To  such  cells  with  their  peculiar  ferments  the  term  "organized 
ferments"  is  often  applied.  The  common  yeast  cell  serves 
as  an  example.  In  other  cases  the  cells  throw  off  in  the 
course  of  their  living  activities,  as  by-products  so  to  speak, 
bodies  which,  when  completely  separated  from  the  cells  by 
which  they  were  formed,  are  still  capable  of  bringing  about 
fermentation  reactions  when  mixed  with  appropriate  sub- 
stances, without  themselves  undergoing  any  demonstrable 
change.  These  are  denominated  "unorganized  ferments" 
or  "enzymes." 

In  the  case  of  the  disease-producing  bacteria  we  have  an 
analogous  state  of  affairs.  We  find  that  the  tissue  changes 
characterizing  disease  are  due  to  poisons  elaborated  by  the 
living  pathogens.  These  poisons  are  generically  known  as 
toxins,  and  it  is  possible,  though  not  certain,  that  in  causing 
disease  their  activities  may  be  in  some  instances  likened  to 


46  BACTERIOLOGY 

those  of  the  enzymes  of  the  non-disease  producing  group, 
while  in  others  this  is  not  the  case.  In  the  case  of  certain 
pathogens,  as  with  the  yeasts  and  certain  saprophytic  bacteria, 
these  toxins — poisons — are  so  bound  up  with  the  protoplasmic 
bodies  of  the  bacteria  that  they  become  effective  as  poisons 
only  on  the  disintegration  of  the  cells  containing  them;  these 
are  the  "endotoxins."  In  other  instances  the  poisons  are 
diffused  through  the  surrounding  medium  in  which  the 
bacteria  are  growing  and  may  readily  be  separated  from 
the  cells  forming  them  by  the  simple  process  of  filtration. 
These  are  the  free  or  "true  toxins." 

At  one  time  there  was  believed  to  be  an  essential  difference 
between  the  "organized"  and  "unorganized"  ferments,  but 
when  in  1897  E.  Buchner  expressed  the  active  ferment  from 
the  yeast  cell,  and  demonstrated  that  this  active  principle, 
"zymase,"  without  the  aid  of  the  living  cell,  is  capable  of 
transforming  sugar  into  aclohol,  just  as  is  done  by  the 
intact  living  yeast  cells,  it  became  manifest  that  the  old 
distinction  between  "organized"  and  "unorganized"  fer- 
ments is  after  all  not  important.  The  "enzyme"  is  the 
active  agent  and  in  so  far  as  the  result  is  concerned  it  matters 
not  if  it  be  tied  up  in  the  body  of  a  cell  or  diffused  freely  in 
the  medium  surrounding  the  cell. 

The  same  may  be  said  with  regard  to  the  analogous 
"endotoxins"  and  "toxins"  elaborated  by  the  pathogenic 
species,  though  it  must  not  be  assumed  that  the  toxins  act 
in  the  same  way  as  do  the  ferments  or  enzymes.  Such 
knowledge  as  we  have  of  the  mechanism  of  certain  toxic 
activities  justifies  the  statement  that  the  poisons  of  some 
pathogenic  bacteria  enter  into  a  destructive  combination 
with  body  cells  for  which  they  have  a  specific  affinity  and 
that  there  and  then  their  activity  ceases;  the  result  being 


FERMENTS,  ENZYMES,   TOXINS  AND  PTOMAINS     47 

that  the  physiological  activities  of  both  the  poison  and  the 
cells  are  destroyed.  Not  so  with  the  enzymes;  they  are 
characterized  by  the  ability  to  bring. about  profound  altera- 
tions in  the  substances  on  which  they  are  acting  without 
they  themselves  being  appreciably  altered  or  diminished  in 
quantity;  just  as  is  seen  with  many  of  the  inorganic  catalysers 
which,  after  having,  by  their  mere  presence,  promoted  con- 
spicuous changes  in  the  substances  surrounding  them,  are 
found  at  the  end  to  have  undergone  little  or  no  loss  in 
amount  and  to  be  of  identically  the  same  composition  as 
at  the  beginning.  As  to  the  way  in  which  enzymes  act 
nothing  definite  can  be  said.  The  problem  has  for  years 
engaged  the  attention  of  competent  investigators  but  up  to 
the  present  there  is  no  final  opinion.  That  they  differ  in 
nature  and  mode  of  operation  the  one  from  the  other 
seems  certain;  the  results  of  their  activities  are  manifestly 
different. 

Neither  enzymes  nor  toxins  have  ever  been  isolated  in 
a  pure  state.  Both  are  assumed  to  be  amorphous  matters 
of  a  protein  nature  and  all  are  recognized  by  that  which 
they  do;  i.  e.,  by  the  reactions  which  they  originate.  They 
are  characterized  for  their  instability,  particularly  is  this 
the  case  with  the  enzymes.  All  have  many  of  the  essential 
characteristics  of  living  matter;  they  are  destroyed  by  heat, 
varying  in  amount  and  mode  of  application.  The  same 
chernicals  that  are  hurtful  to  living  cells  are  likewise,  in  the 
main,  destructive  of  enzymes  and  toxins;  they  are  soluble 
(or  appear  to  be)  in  water,  dilute  acids,  alkalies  and  neutral 
salines;  they  are  to  a  slight  extent  dyalizable;  some  are 
precipitated  from  their  solutions  by  alcohol  readily,  others 
less  so;  they  may  be  thrown  down  from  their  solutions  by 
mechanically  enmeshing  them  with  certain  inorganic 


48  BACTERIOLOGY 

precipitates.    Their  powers  of  fermentation  (enzymes)  and 
of  intoxication  (toxins)  are  apparently  specific. 

The  enzymes  of  bacterial  origin  with  which  we  are  best 
acquainted  may  be  defined  as  amorphous  constituents  of 
living  protoplasm  that  are  able  through  catalytic  activity 
to  split  up  complex  organic  substances  into  simpler,  more 
soluble  and  diffusible  combinations.  They  may  be  classified 
as  proteolytic,  diastatic,  inverting,  coagulating,  sugar 
splitting,  fat  splitting,  etc.  It  is  important  to  note  that 
such  enzymes  may  and  do  originate  in  both  the  animal 
and  vegetable  world.  Those  obtained  from  bacteria  are, 
in  so  far  as  it  is  possible  to  say,  identical  with  those  found 
in  the  cells  of  animals. 

The  proteolytic  or  albumin-dissolving  enzymes  are  formed 
by  a  great  many  bacteria.  The  most  familiar  indications  of 
the  formation  of  a  proteolytic  enzyme  are  seen  in  the  lique- 
faction of  gelatin,  in  the  digestipn  of  coagulated  blood  serum, 
and  of  casein.  Most  frequently  the  proteolytic  enzyme  is 
allied  to  trypsin,  since  the  liquefaction,  hydrolysis  or 
digestion  induced  by  it  proceeds  only  under  an  alkaline  reac- 
tion.1 Some  bacteria,  however,  produce  a  proteolytic  enzyme 
analogous  to  pepsin,  and  this  enzyme  is  active  under  an 
acid  reaction.  The  proteolytic  enzymes  of  different  bacteria 
vary  considerably  with  regard  to  their  resistance  to  heat, 
some  being  destroyed  in  a  few  minutes  when  heated  to  60° 
or  70°  C.,. while  others  may  be  exposed  to  100°  C.  for  a  short 
time  without  suffering  marked  deterioration.2  The  proteo- 
lytic enzymes  also  differ  in  respect  to  their  susceptibility  to 
the  action  of  acids  and  other  chemicals. 

The  formation  of  proteolytic  enzymes  is  one  of  the  func- 

1  See  Abbott  and  Gildersleeve,  Journ.  of  Med.  Research,  1903,  vol.  v. 

2  Loc.  cit. 


FERMENTS,  ENZYMES,   TOXINS  AND  PTOMAINS      49 

tions  of  bacteria  that  is  easily  disturbed  by  external  condi- 
tions, for  instance,  long-continued  cultivation  on  media 
in  which  the  exercise  of  this  function  is  not  required  may 
lead  to  its  marked  deterioration,  while  prolonged  cultivation 
under  conditions  demanding  it  may  result  in  its  accentua- 
tion. 

The  addition  of  carbohydrates  and  of  glycerine  to  culture 
media  interferes  with  production  of  the  proteolytic  enzyme 
by  many  species  of  bacteria,  as  shown  by  Auerbach.1 

Diastatic  enzymes  convert  starch  into  sugar.  This  func- 
tion is  best  studied  on  media  containing  starch,  as  potato 
infusion  or  solutions  of  starch.  By  appropriate  tests  the 
intermediate  steps  in  the  conversion  of  the  starch  into 
sugar  may  be  traced  by  testing  a  portion  of  the  culture 
medium  from  time  to  time.  Fermi2  found  this  function 
in  a  large  number  of  bacteria  studied,  especially  in  organisms 
of  the  subtilis  group  and  in  the  microspira  of  the  cholera 
group. 

Inverting  enzymes  convert  saccharose  into  dextrose  and 
levulose.  These  enzymes  are  produced  by  comparatively 
few  bacteria.  Fermi  found  this  function  manifested  by 
bacillus  megatherium,  pseudoinonas  fluorescens,  bacillus 
vulgaris,  microspira  comma,  microspira  Metchnikovi,  and 
others. 

Coagulating  enzymes  are  those  which  coagulate  milk. 
Rennet  may  be  taken  as  the  typical  form.  This  alteration 
is  quite  common  in  association  with  an  acid  reaction,  but 
in  such  instances  it  is  not  always  certain  that  the  coagulation 
has  not  been  induced  by  the  acid  formed.  Gorini3  found 

1  Archiv  fiir  Hygiene,  Ed.  xxxi,  p.  311. 

2  Ibid.,  Bd.  xi,  and  Centralblatt  fiir  Bacteriologie,  Bd.  xii. 

3  Centralblatt  fur  Bacteriologie,  Bd.  xii,  p.  666. 
4 


50  BACTERIOLOGY 

that  cultures  of  bacillus  prodigiosus,  sterilized  by  heating 
to  60°  C.,  caused  a  solid  coagulation  of  sterile  milk  in  a  few 
days. 

A  small  number  of  bacteria  have  also  been  encountered 
that  bring  about  coagulation  of  milk  with  a  distinctly 
alkaline  reaction.  This  function  has  been  noticed  in  bac- 
teria isolated  from  milk,  and  especially  in  bacterium  pseudo- 
diphtheriticum  isolated  from  cows'  milk  (Bergey). 

Sugar-splitting  enzymes  are  .  very  common  in  bacteria. 
This  function  varies  in  different  species  as  seen  in  the  dif- 
ferent end-products  that  are  formed.  Buchner  succeeded 
in  isolating  the  sugar-splitting  enzyme  (zymase)  of  yeast- 
cells,  and  when  thus  isolated  it  still  possesses  the  power 
of  inducing  active  fermentation  of  sugar.  It  is  believed  that 
the  sugar-splitting  enzymes  of  bacteria  are  similar  in  charac- 
ter to  the  zymase  of  yeast  cells.  The  splitting  up  of  carbo- 
hydrates appears  to  be  brought  about  by  the  bacteria  for 
the  purpose  of  obtaining  oxygen,  as  indicated  by  the  nature 
of  the  end-products  formed,  and  also  by  the  conditions 
under  which  it  may  be  carried  out  —  i.  e.,  the  absence  of 
atmospheric  oxygen. 

The  splitting  of  the  carbohydrate  molecule  may  be  illus- 
trated as  follows: 


=  2C2H6O  +  2CO2 
Grape  sugar  =  2  alcohol  +  2  carbon  dioxide 

or  C6Hi2O6  =  2C3H6O3 

Grape  sugar  =  2  lactic  acid         , 

or  CsHizOe  =  3C2H4p2 

Grape  sugar  =  3  acetic  acid 

According  to  Theobald  Smith1  all  facultative  anaerobic 
bacteria2  form  acids  from  carbohydrates,  while  the  strictly 

1  Centralblatt  fur  Bacteriologie,  Bd.,  xviii. 

2  See  "aerobic"  and  "anaerobic"  bacteria. 


FERMENTS,  ENZYMES,  TOXINS  AND  PTOMAINS      51 

aerobic  bacteria  do  not  have  this  function,  or  bring  about 
the  alteration  so  slowly  that  it  is  concealed  by  the  simul- 
taneous production 'of  alkali.  Among  the  acids  formed  by 
bacteria,  besides  carbon  dioxide,  we  have  lactic,  acetic, 
butyric,  propionic,  and  formic;  and  frequently  there  is 
also  produced  ethyl  alcohol,  aldehyde,  and  acetone. 

The  lactic  acid  formed  by  the  action  of  different  bacteria 
on  carbohydrates  may  be  either  dextrorotatory  or  levoro- 
tatory,  or  almost  equal  quantities  of  both  forms  may  be 
present  and  the  mixture  be  optically  inactive. 

Bacterial  Proteins. — The  proteid  matter  making  up  the 
bodies  of  many  species  of  bacteria,  even  those  not  conspicu- 
ously pathogenic,  was  shown  by  H.  Buchner  to  induce  dis- 
ease when  isolated  and  injected  into  the  tissues  of  animals; 
in  some  cases  causing  only  slight  fever,  in  others  acute 
inflammation  with  suppuration.  For  such  compounds  he 
suggested  the  name  "bacterial  proteins." 

Ptomains. — Ptomains,  or  as  they  are  sometimes  called 
"putrefactive  alkaloids"  or  "cadaveric  alkaloids,"  are 
crystallizable,  nitrogenous  bodies  that  are  the  results  of 
bacterial  action  upon  dead  organic  matter.  They  differ 
from  enzymes  in  that  they  are  the  occasional  results  of 
defective  bacterial  metabolism  and  from  both  toxins  and 
enzymes  in  that  they  are  crystallizable  and  of  definite 
chemical  composition.  Some  of  them  are  poisonous,  many 
are  not.  The  conditions  favorable  to  the  elaboration  of 
ptomains  by  bacteria  vary,  but  in  the  main  the  most 
poisonous  of  the  ptomains  appear  to  be  the  result  of  bac- 
terial activity  under  a  limited  supply  of  oxygen.  Poisonous 
ptomains  are  sometimes  formed  within  the  intestinal  canal 
of  man  either  as  a  result  of  malf ermentation  or  of  interruption 
of  normal  oxidation.  We  have  no  reason  for  believing  that 


52  BACTERIOLOGY 

ptomains  play  any  part  in  either  the  causation  or  course 
of  the  definite,  infective  diseases. 

Ptomains  have  been  isolated  from  decomposing  cadavers, 
from  putrid  meat,  milk,  cheese,  and  from  a  number  of 
bacterial  cultures.  Poisonous  ptomains  occasionally  develop 
in  improperly  preserved  food.  True  toxins  and  dangerous 
bacteria  have  also  been  found  in  such  substances. 

Nutrition  of  Bacteria. — :We  have  said  that  through  the 
agency  of  chlorophyl,  in  the  presence  of  sunlight,  the  green 
plants  are  enabled  to  obtain  the  amount  of  nitrogen  and 
carbon  which  is  necessary  to  their  growth  from  such  simple 
bodies  as  carbon  dioxide  and  ammonia,  which  they  decom- 
pose into  their  elementary  constituents.  The  bacteria,  on 
the  other  hand,  owing  to  the  absence  of  chlorophyl  from 
their  tissues,  do  not  possess  this  power.  They  must,  there- 
fore, have  their  carbon  and  nitrogen  presented  as  such,  in 
the  form  of  decomposable  organic  substances. 

In  general,  bacteria  obtain  their  nitrogen  most  readily 
from  soluble  proteins,  and  to  a  certain  extent,  but  by  no 
means  so  easily,  from  salts  of  ammonium.  In  some  of 
Nageli's  experiments  it  appeared  probable  that  they  could 
obtain  the  necessary  amount  of  nitrogen  from  inorganic 
nitrates.  At  all  events,  he  was  able  in  certain  cases  to 
demonstrate  a  reduction  of  nitric  to  nitrous  acid  and  ulti- 
mately to  ammonia.  Nevertheless,  in  all  of  these  experi- 
ments circumstances  point  to  the  probability  that  the 
nitrogen  obtained  by  the  bacteria  for  building  up  their 
tissues  in  the  course  of  their  development  was  derived  from 
some  source  other  than  the  nitric  acid  or  the  nitrates,  and 
that  the  reduction  of  this  acid  was  most  probably  a  secondary 
phenomenon.  We  must  bear  in  mind,  however,  the  specific 
group,  the.  nitrifying  bacteria,  which  increase  and  mul- 


FERMENTS,  ENZYMES,   TOXINS  AND  PTOMAINS      53 

tiply  without  appropriating  proteid  nutrition.  They  are,  as 
stated  above,  concerned  in  the  particular  form  of  fermenta- 
tion that  results  in  the  oxidation  of  ammonia  to  nitrous  and 
nitric  acids,  a  process  everywhere  in  progress  in  the  super- 
ficial layers  of  the  soil. 

For  the  supply  of  carbon  many  of  the  carbon  compounds 
serve  as  sources  upon  which  the  bacteria  can  draw.  The 
carbon  deficit,  for  example,  can  be  obtained  from  sugar  and 
bodies  of  like  composition;  from  glycerin  and  many  of  the 
fatty  acids;  and  from  the  alkaline  salts  of  tartaric,  citric, 
malic,  lactic,  and  acetic  acids.  In  some  instances  carbon 
compounds,  which  when  present  in  concentrated  form 
inhibit  the  growth  of  bacteria,  may,  when  highly  diluted, 
serve  as  nutrition  for  them.  Salicylic  acid  and  ethyl  alcohol 
are  of  this  class. 

In  addition-  to  carbon  and  nitrogen,  water  is  essential 
to  the  life  and  development  of  bacteria;  without  it  no 
development  occurs,  and  in  many  cases  drying  kills  them. 
Certain  species  and  developmental  forms,  on  the  contrary, 
though  incapable  of  multiplying  when  in  the  dry  state, 
may  be  dried  without  causing  them  to  lose  the  power  of 
reproduction  when  again  placed  under  favorable  conditions. 

Closer  study  of  bacteria,  and  a  more  intimate  acquain- 
tance with  their  nutritive  changes,  demonstrate  an  appre- 
ciable variability  in  the  character  of  the  substances  best 
suited  for  the  nutrition  of  different  species,  as  well  as  in  the 
end  products  of  such  nutrition,  for  instance:  one  species 
may  require  a  tolerably  concentrated  form  of  nutrition, 
while  another  needs  but  a  very  limited  amount  of  proteid 
substance  for  its  development;  some  bring  about  profound 
alterations  in  the  media  in  which  they  are  growing,  while 
others  produce  but  little  apparent  change;  for  certain  species 


54  BACTERIOLOGY 

free  oxygen  is  essential,  for  others  it  is  harmful.  In  one  case 
alterations  in  the  reaction  of  the  media  will  be  conspicuous, 
while  in  another  no  such  variation  can  be  detected.  As 
shown  above  the  growth  of  some  species  is  accompanied  by 
evidence  of  specific  fermentations;  of  others  by  the  appear- 
ance of  poisonous;  of  others  by  putrefactive  changes. 

In  considering  the  normal  development  of  bacteria  we 
must  not  lose  sight  of  the  fact  that  this  is  influenced  both 
by  the  quality  and  the  quantity  of  the  nutritive  materials 
to  which  they  have  access,  and  by  the  character  of  the 
metabolic  products  that  accumulate  in  these  materials  as 
a  result  of  their  vital  processes.  Nitrogen  and  carbon 
compounds  may  be  present  in  amount  and  kind  entirely 
suitable  to  normal  bacterial  growth,  and  yet  this  may  be 
checked,  after  a  comparatively  short  time,  by  the  accumu- 
lated products  of  bacterial  metabolism,  some  of  which 
possess  the  property  of  inhibiting  growth  and  ultimately 
of  even  destroying  the  bacteria  that  produced  them.  The 
most  common  and  conspicuous  examples  of  such  inhibiting 
conditions  is  alteration  in  the  chemical  reaction  of  the  media 
in  which  the  bacteria  are  developing. 

In  the  case  of  a  number  of  species  there  begins,  coincidently 
with  retardation  of  normal  development,  a  process  of  dis- 
solution, self-digestion  or  "autolysis,"  which  may  continue 
until  the  cells  are  unrecognizable  as  bacteria.  This  pheno- 
menon is  the  result  of  the  action  of  enzymes  located  within 
the  cells  which,  under  normal  conditions  of  growth,  are 
concerned  in  the  life  processes  of  the  cell,  but  which,  on  the 
advent  of  conditions  unfavorable  to  the  growth  and  mul- 
tiplication of  the  cells,  .react  upon  them  and  cause  their 
actual  solution.  An  analogous  "autolysis"  is  often  to  be 
seen  with  animal  cells.  If  bits  of  living  tissue  be  removed 


FERMENTS,  ENZYMES,  TOXINS  AND  PTOMAINS      55 

from  the  body,  under  aseptic  precaution,  and  kept  at  suit- 
able conditions  of  moisture  and  temperature  they  may 
ultimately  become  completely  liquefied  as  a  result  of  the 
digestive  action  of  hydrolysing  enzymes  contained  within 
them. 

Their  Relation  to  Oxygen. — Of  primary  importance  and 
interest  in  the  study  of  the  nutritive  changes  of  bacteria 
is  the  difference  in  their  relation  to  oxygen.  For  certain 
species  free  oxygen  is  essential  to  the  proper  performance 
of  their  functions;  in  another  group  no  evidence  of  life  can 
be  detected  under  its  access;  while  in  a  third  group  free 
oxygen  appears  to  play  but  an  unimportant  role,  for  develop- 
ment occurs  as  well  with  as  without  it.  It  was  Pasteur  who 
first  demonstrated  the  existence  of  particular  species  of 
bacteria  which  not  only  grow  and  multiply  and  perform 
definite  physiological  functions  without  the  aid  of  free 
oxygen,  but  to  the  existence  of  which  it  is  positively  harmful. 
To  these  he  gave  the  name  anaerobic  bacteria,  in  contra- 
distinction to  the  aerobic  group,  for  the  proper  performance 
of  whose  functions  free  oxygen  is  essential.  In  addition  to 
these  there  is  a  third  group,  for  the  maintenance  of  whose 
existence  the  absence  or  presence  of  uncombined  oxygen  is 
apparently  of  no  moment — development  progressing  as  well 
with  as  without  it;  the  members  of  this  group  comprise  the 
class  known  as  facultative  in  their  relation  to  this  gas.  It  is 
'to  this  third  group,  the  facultative,  that- the  majority  of 
bacteria  belong. 

It  is  also  well  to  remember  that  many  of  the  so-called 
obligate  anaerobes  may,  by  a  gradual  process  of  adaptation, 
adjust  themselves  to  atmospheres  containing  oxygen.  A 
few  observations  have  shown  that  even  so  anaerobic  a 
species  as  the  bacillus  of  tetanus  may  be  brought  gradually 


56  BACTERIOLOGY 

to  grow  and  perform  all  its  important  functions  in  ordinary 
atmospheric  air. 

Since  all  growing  bacteria,  anaerobic  as  well  as  aerobic, 
generate  carbonic  acid  in  the  course  of  their  development, 
it  is  evident  that  oxygen  must  in  reality  be  obtained  by 
them  from  some  source,  and  must  be  regarded  as  essential 
to  their  life  processes;  but  the  manner  in  which  it  is 
appropriated  by  them  varies,  the  aerobic  species  taking 
it  from  the  air  as  free  oxygen,  while  the  anaerobic  species, 
not  possessed  of  this  ability,  obtain  it  through  the  decom- 
position of  more  or  less  stable  oxygen-containing  com- 
pounds. 

Though  the  multiplication  of  the  facultative  varieties  is 
not  interfered  with  by  either  the  presence  or  absence  of  free 
oxygen,  yet  experiments  demonstrate  that  the  products  of 
their  growth  are  different  under  the  varying  conditions  of 
absence  or  presence  of  this  gas.  For  example:  in  the  case' 
of  certain  of  the  chromogenic  forms  the  presence  or  absence 
of  oxygen  has  a  very  decided  effect  upon  the  production  of 
the  pigments  by  which  they  are  characterized. 

NOTE. — Observe  the  difference  between  the  intensity  of 
color  produced  upon  the  surface  of  the  medium  and  that 
along  the  track  of  the  needle  in  stab-cultures  of  bacillus 
prodigiosus  and  of  spirillum  rubrum.  In  the  former  the  red 
color  is  apparently  a  product  dependent  upon  the  presence 
of  oxygen,  while  in  the  latter  the  greatest  intensity  of  color 
occurs  at  the  point  furthest  removed  from  the  action  of 
oxygen. 

Influence  of  Temperature  upon  the  Growth. — Another 
factor  which  plays  a  highly  important  part  in  the  biological 


FERMENTS,  ENZYMES,   TOXINS  AND  PTOMAINS       57 

functions  of  these  organisms  is  the  temperature  under  which 
they  exist.  The  extremes  of  temperature  between  which 
the  majority  of  bacteria  are  known  to  grow  range  from  5.5° 
to  43°  C.  At  the  former  temperature  development  is  hardly 
appreciable;  it  becomes  more  and  more  active  until  38°  C. 
is  reached,  when  it  is  at  its  optimum,  and,  as  a  rule,  ceases 
at  43°  C.;  though  species  exist  that  multiply  at  as  high  a 
temperature  as  70°  C.  and  others  at  as  low  as  0°  C.  The 
investigations  of  Globig,1  Miquel,2  and  Macfayden  and 
Bloxall3  have  revealed  the  existence  in  the  soil,  in  water, 
in  feces,  in  sewage,  in  dust,  and,  in  fact,  practically  every- 
where, of  bacteria  that  under  artificial  cultivation  show  no 
evidence  of  life  at  a  temperature  lower  than  60°  to  65°  C., 
and  will  even  grow  at  such  high  temperatures  as  70°  and 
75°  C.,  a  state  of  affairs  almost  paradoxical,  inasmuch  as 
these  are  temperatures  that  suffice  for  the  coagulation  of 
albumin,  and,  in  consequence,  are  generally  incompatible 
with  life.  Rabinowitsch4  has  likewise  described  a  number 
of  species  of  these  thermophilic  bacteria,  as  they  are  called; 
but  states  that  it  was  possible  in  her  experiments  to  obtain 
evidence  of  their  growth  at  the  lower  temperature  (34°  to 
44°  C.),  as  well  as  at  the  higher  temperature  mentioned  by 
preceding  investigators.  It  is  possible  that  this  peculiarity 
is  but  a  manifestation  of  adaptation  to  environment  and 
not  an  essential  to  the  life  processes  of  these  species. 

The  most  favorable  temperature  for  the  development  of 
.pathogenic  bacteria  is  that  of  the  human  body,  viz.,  37.5°  C. 
There  are  a  number  of  bacteria  commonly  present  in  water, 

1  Zeitschrift  fur  Hygiene,  Bd.  iii,  S.  294. 

2  Annales  de  Micrographie,  1888,  pp.  4  to  10. 

3  Journal  of  Path,  and  Bact.,  vol.  iii,  Part  I. 

4  Zeitschrift  fur  Hygiene  u.   Infecktionskrankheiten,   Bd.   xx,   Heft.    1, 
S.  154  to  164. 


58  BACTERIOLOGY 

the  so-called  normal  water  bacteria,  that  grow  best  at  about 
20°  C. 

Reaction. — The  majority  of  bacteria  require  an  approx- 
imately neutral  medium  in  which  to  multiply  and  function. 
Certain  species  may  develop  in  weak  acid  materials,  others 
in  weak  alkaline — but  none  can  live  and  grow  in  media 
either  strongly  acid  or  alkaline.  Even  those  species  whose 
most  conspicuous  function  is  the  conversion  of  sugars  into 
acids  have  their  activities  checked  by  the  accumulation  of 
free  acid  beyond  a  very  limited  amount. 

Cooperating  Bacteria. — Under  natural  conditions  it  fre- 
quently occurs  that  the  development  of  one  species  or  group 
of  species  of  bacteria  is  directly  dependent  upon  the  func- 
tional activities  of  another  totally  distinct  species,  the 
growth  of  one  group  resulting  in  conditions  that  are  of  vital 
importance  to  the  existence  of  the  other.  Such  interdepen- 
dence is  observed,  for  instance,  in  complete  nitrification, 
as  already  noted;  in  the  course  of  putrefaction,  where, 
through  exhaustion  of  free  oxygen  by  the  actively  germinat- 
ing aerobic  varieties,  the  conditions  are  supplied  that  enable 
the  anaerobic  species  to  develop  and  exercise  their  biological 
activities.  Again,  through  the  proteolytic  activity  of 
enzymes  produced  by  certain  species  of  bacteria,  other 
species  are  supplied  with  nutrition  that  would  otherwise  be 
unassimilable  or  only  imperfectly  so.  Similar  cooperative 
or  symbiotic  relations  between  bacteria  and  higher  plants 
are  also  noticed,  notably  that  between  certain  bacteria  of 
the  soil  and  the  group  of  leguminous  plants,  whereby  the 
latter  are  enabled,  through  the  assistance  of  the  former,  to 
make  up  their  nitrogen  deficit  in  large  part  from  the  free 
nitrogen  of  the  atmosphere.  This  latter  relationship  is 
probably  an  example  of  true  symbiosis.1 

1  See  Nitrogen  fixing  bacteria. 


FERMENTS,  ENZYMES,   TOXINS  AND    PTOMAINS     59 

Influence'  of  Light. — Light  is  not  only  unnecessary  to  the 
performance  of  functions  by  bacteria  but  appears  to  be 
in  varying  degrees  inhibitory. 

•  Direct  sunlight  is  destructive  to  many  species.  It  is  a 
matter  of  common  experience  that  cultures  of  particularly 
important  species  retain  their  type  characteristics  better 
and  longer  if  cultivated  in  the  dark  than  in  diffuse  daylight. 

Electric  light  has  likewise  a  depressing  influence  upon 
the  viability  of  bacteria.  Beyond  the  fact  that  bacteria  in 
vacuo  are  unaffected  by  light  we  have  no  knowledge  of  the 
mechanism  of  its  action.  Presumably  it  has  something  to 
do  with  oxidation  processes. 

The  germicidal  action  of  the  direct  rays  of  the  sun  may 
be  easily  demonstrated  by  preparing  a  plate  of  colon  bacillus, 
shading  a  portion  and  allowing  the  sun  to  shine  upon  it  for 
a  time,  varying  with  the  intensity  of  its  light.  Growth  will 
occur  in  the  shaded  part,  none  or  only  relatively  little  in 
the  illuminated  part  of  the  plate. 

Influence  of  Pressure. — The  influence  of  pneumatic  pres- 
sure on  the  viability  of  bacteria  appears  to  depend  upon  the 
character  of  the  gas  used.  Ordinary  air,  or  its  constituents, 
oxygen  and  nitrogen,  whenever  pressed  heavily  (600  to 
2000  atmospheres)  upon  cultures  of  bacteria,  have  a  slight 
inhibitory  effect;  Carbon  dioxide  under  five  to  ten  atmos- 
pheres pressure  is  shown  by  Park  and  his  associates  to 
destroy  almost  all  of  the  typhoid,  dysentery,  diphtheria 
and  colon  bacilli  exposed  to  it  within  twenty-four 
hours. 

Effect  of  Moisture. — As  is  the  case  with  all  living  plants 
a  degree  of  moisture  is  essential  to  life.  Certain  species  of 
bacteria  are  killed  by  ordinary  drying,  and  many  of  them  by 
absolute  drying.  The  spores  (to  be  described  later)  of  bac- 


60  BACTERIOLOGY 

teria  are  not  so  effected,  a  few  species  retaining  their  power 
to  germinate  after  having  been  dried,  as  the  word  is  ordinarily 
understood,  for  a  comparatively  long  time,  and  spores  have 
been  kept  in  a  dry  state  for  years  without  losing  their  power 
to  germinate. 

Influence  of  Electricity. — The  methods  employed  for 
deciding  this  point  have  led  to  results  that  are  inconclusive 
and  not  easy  of  interpretation. 

It  is  true  that  when  bacteria  are  exposed  to  the  electric 
current  they  are  often  inhibited  and  sometimes  killed. 

This  result  may  be  interpreted  in  several  ways,  viz. :  The 
elevation  of  temperature  caused  by  the  current  may  explain 
the  destruction;  the  electrolytic  action  of  the  current  on 
matters  in  which  the  bacteria  are  located  may,  by  dissocia- 
tion, liberate  agents  that  are  destructive  to  bacteria,  or  a 
similar  destructive  dissociation  within  the  bacteria  them- 
selves may  result  from  the  action  of  the  current. 

The  evidence  at  hand  does  not  permit  of  the  acceptance 
of  either  of  these  suggestions  as  the  correct  interpretation  of 
the  results. 

Chemotaxis. — Another  interesting  biological  peculiarity  of 
bacteria  is  that  discovered  by  Engelmann  and  by  Pfeffer, 
known  as  chemotaxis.  This  term  applies  to  the  peculiar 
phenomena  of  attraction  and  of  repulsion  that  are  exhibited 
by  motile  bacteria  when  in  the  presence  of  solutions  of  bodies 
of  various  chemical  composition.  It  was  demonstrated 
that  the  bacteria  in  decomposing  infusions  accumulate  in 
great  numbers  in  the  neighborhood  of  the  sources  of  oxygen. 
In  a  hanging-drop  of  such  an  infusion  the  bacteria  will  be 
seen  to  a'ccumulate  in  a  dense  mass  along  the  margin  or 
around  the  edge  of  small  bubbles  of  air  in  the  fluid.  Even 


FERMENTS,  ENZYMES,   TOXINS  AND  PTOMAINS      61 

plant  cells  in  the  infusion,  whose  chlorophyl  sets  free  oxygen 
in  the  light,  are  surrounded  by  large  numbers  of  bacteria. 
The  positive  chemotactic  affinity  between  oxygen  and 
bacteria  was  employed  by  Engelmann  as  a  basis  for  the 
demonstration  of  small  quantities  of  oxygen  in  studying  the 
influence  of  various  kinds  of  light  upon  the  assimilation  of 
green  plant-cell.  Pfeffer  showed  that  when  a  neutral  fluid 
(a  drop  of  water)  containing  motile  bacteria  is  brought  in 
contact  with  a  weak  solution  of  either  peptone,  sodium 
chloride,  or  dextrin,  the  bacteria  are  at  once  attracted 
toward  the  solution;  this  reaction  is  designated  "positive 
chemotaxis."  On  the  other  hand,  if  brought  in  contact  with 
an  acid,  an  alkaline,  or  an  alcoholic  solution,  the  bacteria 
are  repelled  or  driven  from  the  point  at  which  the  two  fluids 
are  diffusing;  that  is,  they  exhibit  "negative  chemotactic" 
affinities.  The  significance  of  these  reactions  is  not  under- 
stood, but  it  has  been  aptly  suggested  that  they  may  be 
fundamentally  analogous  to  the  specific  positive  and  negative 
affinities  exhibited  by  the  ions  resulting  from  dissociation 
of  electrolytes,  and  that  they  may  "have  their  explanation 
in  the  forces  of  ionic  attraction  and  repulsion."1  In  this 
connection  it  is  important  to  note  that  the  wandering 
cells  of  the  animal  body,  the  leukocytes,  exhibit  also  these 
chemotactic  phenomena;  and  it  is-  especially  necessary  to 
a  complete  comprehension  of  the  process  of  suppuration  to 
bear  in  mind  that  among  the  substances  which  have  the 
greatest  attraction  for  these  wandering  cells,  are  the  products 
of  growth  of  certain  bacteria  in  some  cases,  and  the  protein 
constituents  of  the  bacteria  themselves  in  others. 


1  Read  Sewall  on  Some  Relations  of  Osmosis  and  Ionic  Action  in  Clinical 
Medicine,  International  Clinics,  vol.  xi,  Eleventh  Series. 


62  /     BACTERIOLOGY 

To  summarize  briefly  the  foregoing  it  may  be  said,  in 
general,  that  for  the  growth  and  development  of  bacteria 
nitrogenous  organic  matter  of  a  neutral  or  slightly  alkaline 
reaction,  in  the  presence  of  moisture  and  at  a  suitable  tem- 
perature, is  all  that  is  necessary.  From  this  can  be  formed 
some  idea  of  the  omnipresence  in  nature  of  these  minute 
vegetables.  Bacteria  are  found  wherever  these  conditions 
obtain. 


CHAPTER  II. 

Morphology1   of  Bacteria — Chemical   Composition  of  Bacteria — Mode   of 
Multiplication — Spore-formation — Motility. 

IN  structure  the  bacteria  are  unicellular,  always  develop- 
ing from  preexisting  cells  of  the  same  character  and  never 
appearing  spontaneously.  They  are  seen  to  occur  as  spher- 
ical, rod-  and  spiral-shaped  bodies  that  multiply  by  the 
simple  process  of  transverse  division,  belonging,  therefore,  to 
the  schizomycetes  or  fission  fungi. 

In  size  the  bacteria  are  among  the  smallest  living  crea- 
tures with  which  we  are  acquainted,  being  visible  only 
when  very  highly  magnified.  In  order  that  some  conception 
of  their  microscopic  dimensions  may  be  formed,  it  has  been 
computed  that  of  the  average  size  bacteria  about  thirty 
billion  would  be  required  to  weigh  a  gram,  and  that  about 
one  billion  seven  hundred  million  of  the  small  spherical  forms 
might  readily  be  suspended  in  a  drop  of  water. 

Under  what  we  are  accustomed  to  regard  as  normal  con- 
ditions of  development,  and  by  the  ordinary  methods  of 
examination,  bacteria  appear  very  simple  in  form  and 
structure.  They  are  cells  consisting  of  a  protoplasmic  mass 
within  a  membranous  hull  that  is  discernible  with  more  or 
less  difficulty.  The  protoplasmic  body  is  of  material  closely 
allied,  chemically  speaking,  to  ordinary  vegetable  protein. 
It  is  often  homogenous,  but  in  particular  species  and  under 
various  conditions  of  growth  the  central  mass  in  stained 

1  Morphology,  pertaining  to  shape,  outline,  structure. 

(63) 


64  BACTERIOLOGY 

specimens  is  commonly  marked  by  the  presence  of  very 
dark  granules,  the  so-called  metachromatic  granulations. 
Again,  in  other  species  paraplastic  granules  giving  the 
microchemical  reactions  of  fat,  starch,  sulphur,  etc.,  are  to 
be  seen.  Under  certain  physical  conditions  the  protoplasmic 
body  presents  irregular  rents  or  retractions,  the  result  of 
proteolytic  or  of  osmotic  disturbances  dependent  upon  the 
character  of  the  fluid  in  which  the  bacteria  are  located;  in 
fact,  the  deeply  staining  granules,  other  than  those  of  fat, 
starch,  and  sulphur,  that  are  often  observed,  are  regarded 
by  some  writers  (especially  A.  Fischer)  as  but  altered  or 
condensed  protoplasm  due  to  the  same  influences. 

In  certain  species  the  protoplasmic  body  is  always  more 
dense  at  the  poles  of  the  cells  than  at  the  middle,  so  that 
when  stained  the  ends  are  much  darker  than  the  intervening 
portion.  In  other  species  the  reverse  is  the  case. 

By  some  investigators  the  protoplasmic  central  mass  is 
regarded  as  a  nucleus,  and,  functionally  speaking,  possibly 
it  is  to  all  intents  and  purposes,  but  this  cannot  be  certainly 
decided.  In  the  great  majority  of  cases,  however,  with  the 
ordinary  methods  of  examination,  it  is  not  seen  to  possess 
any  of  the  structural  peculiarities  that  we  are  accustomed 
to  regard  as  the  distinguishing  attributes  of  cell-nuclei. 

The  enveloping  hull  or  membrane  is  in  some  cases  ap- 
parently only  a  modification  of  the  protoplasmic  central 
mass,  at  times  being  only  a  condensation  of  that  protoplasm; 
again,  it  seems  to  be, chemically  different  from  it.  In  a  few 
instances  it  appears  to  be  allied  to  cellulose  in  its  chemical 
composition.  Sometimes  it  is  so  thick  as  to  be  readily  seen, 
while  again  it  is  discernible  only  by  special  methods  of 
examination.  In  particular  species  it  may,  by  appropriate 
methods,  be  seen  as  a  sharply  defined  capsule  inclosing  a 


CHEMICAL  COMPOSITION  OF  BACTERIA  65 

clear  zone  in  which  lies  the  deeply  stained  central  mass. 
Occasionally  the  central  protoplasmic  mass  is  surrounded 
by  an  ill-defined  slimy  material  that  causes  the  individual 
cells  to  adhere  to  one  another  in  more  or  less  compact  masses 
or  pellicles  (zooglea,  Fig.  1). 

Chemical  Composition  of  Bacteria. — The  bodies  of  bacteria 
consist  of  water,  salts,  and  albuminous  substances,  with 
smaller  proportions  of  various  extractives  soluble  in  alcohol 
or  ether,  such  as  triolein,  tripalmitin,  tristearin,  lecithin, 
and  cholesterin.  In  many  varieties  substances  giving  the 
reaction  of  starch  have  been  found,  while  others  give  the 
true  reactions  of  cellulose  (B.  subtilis).  Nuclein  has  not 
* 

FIG.  1 


/  ^>"  '¥•--«&*>.  ?£H.  \ 


!*S£5*3B?.          ,. 

Zooglea  of  bacilli. 

been  found  in  any  of  the  bacteria,  though  the  nuclein  bases, 
xanthin,  guanin,  adenin,  have  been  found. 

The  relative  amounts  of  water  in  bacteria  are  influenced 
to  a  large  extent  by  the  nature  of  the  medium  on  which 
they  have  been  grown.  In  like  manner  the  content  in 
albumin,  extractive  substances,  and  salts  varies  with  the 
conditions  under  which  the  bacteria  have  been  cultivated. 
E.  Cramer1  has  studied  the  chemical  composition  of  bacteria 
in  great  detail.  As  the  result  of  his  studies  of  microspira 
comma,  he  found  its  composition  to  be  as  follows:  water 

1  Archiv  fur  Hygiene,  Bd.  xiii,  xvi,  xxii,  and  xxviii. 


66  BACTERIOLOGY 

88.3  per  cent.,  albumin  7.6  per  cent.,  ash  3.6  per  cent.  The 
dry  substance  of  the  bacteria  contains  the  following :  albumin 
65  per  cent.,  ash  31  per  cent.  From  76  to  80  per  cent,  of 
the  ash  consists  of  sodium  chloride  and  phosphate. 

Morphology  of  Bacteria. — For  the  purposes  of  this  book  it 
will  suffice  to  classify  the  bacteria  roughly  into  three  mor- 
phological groups  with  their  subdivisions,  the  members  of 
each  group  being  identified  by  their  individual  outline,  viz., 
that  of  a  sphere,  a  rod,  or  a  spiral.1  To  these  three  grand 

FIG.  2 


c  d  e 

a,  staphylococci;   b,  streptococci;   c,  diplococci;  d,  tetrads;  e,  sarcinse. 

divisions  are  given  the  names  cocci  or  micrococci,  bacilli, 
and  spirilla. 

Mode  of  Multiplication. — In  the  group  micrococci  belong 
all  spherical  forms — i.  e.,  all  those  forms  the  isolated  individ- 
ual members  of  which  are  practically  of  the  same  diameter 
in  all  directions.  (See  Fig.  2,  a,  6,  c,  d,  e.) 

The  bacilli  comprise  all  oval  or  rod-formed  bacteria.  (See 
Fig.  3.) 

1  For  complete  data  on  classification  see  Reports  of  Committee  of  Am. 
Bact.  Soc.,  Jour.  Bact.,  1917,  vol.  ii;  1920,  vol.  iii. 


MODE  OF  MULTIPLICATION  67 

To  the  spirilla  belong  the  bacteria  that  are  curved  when 
seen  in  short  segments  and  that  appear  as  undulating 
threads  when  such  segments  are  of  greater  length  or  when 
several  short  segments  are  joined  end  to  end.  (See  Fig.  4.) 

FIG.  3 


d  e  f 

a,  bacilli  in  pairs;    b,  single  bacilli;    c  and  d,  bacilli  in  threads;    e  and  /, 
bacilli  of  variable  morphology. 

The  micrococci  are  subdivided  according  to  their  pre- 
vailing mode  of  grouping,  as  seen  in  growing  cultures,  into 
staphylococci — those  growing  in  masses  like  clusters  of  grapes 

FIG.  4 


a  b  c 

a  and  c,  spirilla  in  short  segments  and  longer  threads — the  so-called  comma 

forms  and  spirals;   6,  the  thick  spirals,  known  as  vibrios. 

(see  Fig.  2,  a);  streptococci,  those  growing  in  chains  con- 
sisting of  a  number  of  individuals  strung  together  like 
beads  upon  a  string  (see  Fig.  2,  b) ;  diplococci — those  growing 


68  BACTERIOLOGY 

in  pairs  (Fig.  2,  c);  tetrads — those  developing  as  fours  (Fig. 
2,  d);  and  sarcince — those  dividing  into  fours,  eights,  etc., 
as  cubes — that  is,  in  contradistinction  to  all  other  forms,  the 
segmentation,  which  is  rarely  complete,  takes  place  regularly 
in  three  directions  of  space,  so  that  when  growing  the  bundle 
of  segmenting  cells  presents  somewhat  the  appearance  of  a 
bale  of  cotton  (Fig.  2,  e). 

To  the  bacilli  belong  all  straight,  oval  and  rod-shaped 
bacteria — L  e.,  those  in  which  one  diameter  is  always  greater 
than  the  other.  In  this  group  are  found  those  organisms  the 
life-cycle  of  many  of  which  presents  deviations  from  the 
simple  rod  shape.  Many  of  them  in  the  course  of  development 
increase  in  length  into  long  threads,  along  which  traces  of 
segmentation  may  usually  be  found.  Again,  under  certain 
conditions,  many  of  them  possess  the  property  of  forming 
within  the  body  of  the  rods  oval,  glistening  spores  (see  Fig.  6), 
and,  if  the  conditions  are  not  altered,  the  rods  may  entirely 
disappear  and  nothing  be  left  in  the  culture  but  these  oval 
spores.  In  some  of  them  this  phenomenon  of  spore-formation 
is  accompanied  by  an  enlargement  or  swelling  of  the  bacillus 
at  the  point  at  which  the  spore  is  located  (see  Fig.  6,  c  and  d) . 
Again,  many  of  them,  from  unfavorable  conditions  of  nutri- 
tion, aeration,  or  temperature,  undergo  pathological  changes 
that  are  probably  autolytic  in  nature — that  is,  the  individ- 
uals themselves  experience  degeneration  of  their  proto- 
plasm with  coincident  distortion  of  their  outline;  they  are 
then  usually  referred  to  as  "involution-forms"  (see  Fig.  5, 
a  and  &).  In  all  of  these  conditions,  however,  so  long  as 
death  has  not  occurred,  it  is  possible  to  cause  these  forms 
to  revert  to  the  typical  rods  from  which  they  originated, 
by  the  renewal  of  conditions  favorable  to  their  normajj 
vegetation. 


MODE  OF  MULTIPLICATION  69 

It  must  be  borne  in  mind,  though,  that  it  is  never  possible 
by  any  means  to  bring  about  changes  in  these  organisms 
that  will  result  in  the  permanent  conversion  of  the  mor- 
phology of  the  members  of  one  group  into  that  of  another — 
that  is,  one  can  never  produce  bacilli  from  micrococci,  nor 
vice  versa;  and  any  evidence  which  may  be  presented  to 
the  contrary  is  based  upon  untrustworthy  methods  of 
experimentation. 

Very  short  oval  bacilli  may  sometimes  be  mistaken  for 
micrococci,  and  at  times  micrococci  in  the  stage  of  segmenta- 
tion into  diplococci  may  be  mistaken  for  short  bacilli;  but 


FIG.  5 


tf  \\)  '|/| 

*//» 


.  a,  spirillum  of  Asiatic  cholera  (comma  bacillus) ;  normal  appearance 
in  fresh  cultures;  b,  involution-forms  of  this  organism  as  seen  in  old  cul- 
tures. 


by  careful  inspection  it  will  always  be  possible  to  detect  a 
continuous  outline  along  the  sides  of  the  former,  and  a 
slight  transverse  indentation  or  partition-formation  between 
the  segments  of  the  latter.  The  high  index  of  refraction  of 
spdres,  the  property  which  gives  to  them  their  glistening 
appearance,  will  always  serve  to  distinguish  them  from 
micrococci.  This  difference  in  refraction  is  especially  notice- 
able if  the  illumination  of  the  microscope  be  reduced  to  the 
smallest  possible  bundle  of  light-rays. .  The  spores,  more- 
over, take  up  staining-reagents  much  less  readily  than 
do  the  micrococci.  The  most  reliable  differential  points, 


70  BACTERIOLOGY 

however,  are  the  infallible  properties  possessed4  by  the 
spores  of  developing  into  bacilli,  and  by  the  spherical 
organism  with  which  they  may  have  been  confounded  of 
always  producing  other  micrococci  of  the  same  spherical 
form. 

We  have  less  knowledge  of  the  life-history  of  the  spiral 
forms.  Efforts  toward  their  cultivation  under  artificial 
conditions  have  thus  far  been  successful  in  only  a  compara- 
tively limited  number  of  cases.  Morphologically,  they  are 
thread-  or  rod-like  bodies  which  are  twisted  into  the  form 
of  spirals.  In  some  of  them  the  turns  of  the  spiral  are  long, 
in  others  quite  short.  In  some  the  threads  appear  rigid,  in 
others  flexible.  They  are  motile  and  multiply  apparently  by 
the  simple  process  of  fission.1 

Mode  of  Multiplication. — The  micrococci  multiply  by 
simple  fission.  When  development  is  in  progress  a  single 
cell  will  be  seen  to  elongate  slightly  in  one  of  its  diameters. 
Over  the  center  of  the  long  axis  thus  formed  will  appear  a 
slight  indentation  in  the  outer  envelope  of  the  cell;  this 
indentation  will  increase  in  extent  until  there  exist  even- 
tually two  individuals  which  are  distinctly  spherical,  as  was 
the  parent  from  which  they  sprang,  or  they  will  remain 
together  for  a  time  as  diplococci;  the  surfaces  now  in  juxta- 
position are  flattened  against  one  another,  and  not  infre- 
quently a  fine,  pale  dividing-line  may  be  seen  between  the 
two  cells.  (See  Fig.  2,  c  and  d.)  A  similar  division  in  the 
other  direction  will  now  result  in  the  formation  of  fours  as 
tetrads. 

In  the  formation  of  staphylococci  such  division  occur 
irregularly  in  all  directions,  resulting  in  the  production  of 

1  Dividing  into  two  transversely. 


MODE  OF  MULTIPLICATION  71 

the  clusters  in  which  these  organisms  are  commonly  seen. 
(See  Fig.  2,  a.)  With  the  streptococci,  however,  the  ten- 
dency is  for  the  segmentation  to  continue  in  one  direction 
only,  resulting  in  the  production  of  long  chains  of  4,  8,  and 
12  individuals.  (See  Fig.  2,  6.) 

The  sarcinse  divide  more  or  less  regularly  in  three  direc- 
tions of  space;  but  instead  of  becoming  separated  the  one 
from  the  other  as  single  cells,  the  tendency  is  for  the  seg- 
mentation to  be  incomplete,  the  cells  remaining  together 
in  masses.  The  indentations  upon  these  masses  or  cubes, 
which  indicate  the  point  of  incomplete  fission,  give  to  the 
bundles  of  cells  the  appearance  commonly  ascribed  to  them, 
viz.,  that  of  a  bale  of  cotton  or  a  packet  of  rags.  (See  Fig. 
2>e.) 

The  mode  of  multiplication  of  bacilli  is  similar  to  that 
of  the  micrococci — i.  e.,  a  dividing  cell  elongates  slightly  in 
the  direction  of  its  long  axis;  an  indentation  appears  about 
midway  between  its  poles,  and  this  becomes  deeper  and 
deeper,  until  eventually  two  daughter-cells  have  formed. 
This  process  may  occur  in  such  a  way  that  the  two  young 
bacilli  adhere  together  by  their  adjacent  ends  in  much  the 
same  way  that  sausages  are  seen  to  be  held  together  in 
strings  (Fig.  3,  /),  or  the  segmentation  may  take  place  more 
at  right  angles  to  the  long  axis,  so  that  the  proximal  ends 
of  the  young  cells  are  flattened,  while  the  distal  extremities 
may  be  rounded  or  slightly  pointed  (Fig.  3,  e).  The  segmen- 
tation of  the  anthrax  bacillus,  with  which  we  are  to  become 
acquainted  later,  results,  when  completed,  in  an  indenta- 
tion of  the  adjacent  extremities  of  the  young  segments,  so 
that  by  the  aid  of  high  magnifying  powers  these  surfaces 
are  seen  to  be  actually  concave.  Bacilli  never  divide  longi- 
tudinally. 


72  BACTERIOLOGY 

Spore  Formation. — With  the  spore-forming  bacilli,  under 
favorable  conditions  of  nutrition  and  temperature,  the 
same  mode  of  segmentation  is  seen  to  occur  during  vege- 
tation; but  as  soon  as  these  conditions  become  altered  by 
the  exhaustion  of  nourishment,  the  presence  of  detrimental 
substances,  unfavorable  temperatures,  etc.,  they  enter,  in 
their  life-cycle,  the  stage  to  which  we  have  referred  as 
spore-formation.  This  is  the  process  by  which  the  organisms 
are  enabled  to  enter  a  state  in  which  they  resist  deleterious 
influences  to  a  much  higher  degree  than  is  possible  for  them 
when  in  the  growing  or  vegetative  condition. 

In  the  spore,  dormant,  or  permanent  state,  as  it  is  variously 
called,  no  evidence  of  life  whatever  is  given  by  the  spores; 
though  as  soon  as  the  conditions  which  favor  their  germina- 
tion have  been  renewed  these  spores  develop  again  into  the 
same  kind  of  cells  as  those  from  which  they  originated,  and 
the  appearances  observed  in  the  vegetative  or  growing  stage 
of  their  history  are  repeated. 

Multiplication  of  spores,  as  such,  does  not  occur ;~  they 
possess  only  the  power  of  developing  into  individual  rods 
of  the  same  nature  as  those  from  which  they  were  formed, 
but  not  of  giving  rise  to  a  direct  reproduction  of  spores. 

When  the  conditions  which  favor  spore-formation  present, 
the  protoplasm  of  the  vegetative  cells  is  seen  to  undergo  a 
change.  It  loses  its  normal  homogeneous  appearance  and 
becomes  marked  by  granular,  refractive  points  of  irregular 
shape  and  size.  These  eventually  coalesce,  leaving  the 
remainder  of  the  cell  clear  and  transparent.  When  this 
coalescence  of  highly  refractive  particles  is  complete  the 
spore  is  perfected.  In  appearance  the  spore  is  oval  or  round, 
and  very  highly  refractive — glistening.  It  is  easily  differen- 
tiated from  the  remainder  of  the  cell,  which  now  consists 


SPORE  FORMATION  73 

only  of  a  cell-membrane  and  a  transparent,  clear  space 
which  surrounds  the  spore.  Eventually  both  the  cell- 
membrane  and  its  fluid  contents  disappear,  leaving  the  oval 
spore  free;  it  then  gives  the  impression  of  being  surrounded 
by  a  dark,  sharply  defined  border.  When  thus  perfectly 
developed,  the  spore  may  be  regarded  as  analogous  to  the 
seeds  of  higher  plants.  Like  the  seed,  it  evinces  no  evidence 
of  life  until  placed  under  conditions  favorable  to  germina- 
tion, when  there  develops  from  it  a  cell  identical  in  all 
respects  with  that  from  which  it  originated.  Its  tenacity 
of  life,  as  in  the  case  of  seeds,  is  almost  unlimited.  It  may 

FIG.  6 


a,  Bacillus  subtilis  with  spores;  6,  bacillus  anthracis  with  spores;  c,  clos- 
tridium  form  with  spores;   d,  bacillus  of  tetanus  with  end  spores. 


be  kept  in  a  dry  state,  and  this  has  actually  been  done,  for 
years  without  loss  of  viability. 

The  glistening,  enveloping  spore-membrane  is  not  of 
uniform  thickness  throughout,  and  in  consequence  when 
germination  occurs  the  growing  bacillus,  the  so-called  vege- 
tative form  of  the  organism,  protrudes  through  the  thinnest 
part  of  the  spore-membrane — that  is,  through  the  point  of 
least  resistance.  This  may  be  either  the  end  or  the  side  of 
the  spore,  according  to  the  species  under  observation.  In 
certain  cases  such  a  protrusion  is  not  observed,  but  in  its 
place  the  spore  in  toto  appears  to  be  gradually  absorbed  or 


74  BACTERIOLOGY 

in  some  way  converted  directly  into  a  vegetating  cell.  It 
evinces  no  motion  other  than  the  mechanical  tremor  common 
to  all  insoluble  microscopic  particles  suspended  in  fluids, 
and  it  remains  quiescent  until  there  appear  conditions  favor- 
able to  its  subsequent  development. 

By  the  ordinary  methods  of  staining,  spores  do  not  become 
colored,  so  that  they  appear  in  the  stained  cells  as  pale, 
transparent,  oval  bodies,  surrounded  by  the  remainder  of 
the  cell,  which  has  taken  up  the  dye. 

A  single  cell  produces  but  one  spore.  This  may  be  located 
either  at  an  extremity  or  in  the  center  of  the  cell  (Fig.  6). 

Occasionally  spore-formation  is  accompanied  by  an  en- 
largement of  the  cell  at  the  point  at  which  the  process  is 
in  progress.  As  a  result,  the  cell  loses  its  regular  rod 
shape  and  becomes  that  of  a  club,  a  drum-stick,  or  a  loz- 
enge, depending  upon  whether  the  location  of  the  spore 
is  to  be  at  the  pole  or  in  the  center  of  the  cell.  (See 
Fig.  6,  c  and  d.) 

Motility. — In  addition  to  the  property  of  spore-formation 
there  is  another  striking  difference  between  various  species 
of  bacteria,  namely,  the  property  of  motility,  by  which  some 
of  them  are  distinguished.  This  power  of  motion  is  due  to 
very  delicate,  hair-like  appendages  or  flagella,  by  the  lashing 
motions  of  which  the  cells  possessing  them  are  propelled 
through  the  fluid.  In  some  cases  the  flagella  are  located  at 
but  one  end  of  the  organism,  either  singly  (monotrichic)  or 
in  a  tuft  (lophotrichic) ;  and  in  some  cases,  especially  of 
the  bacillus  of  typhoid  fever,  they  are  given  off  from  the 
whole  surface  of  the  rod  (peritrichic).  (See  Fig.  7.) 

For  a  long  time  this  property  of  independent  motion  could 
only  be  assumed  to  be  due  to  the  possession  of  some  such 
form  of  locomotive  apparatus,  because  similar  appendages 


MOTILITY 


75 


had  been  seen  upon  some  of  the  large  motile  spirilla  found 
in  stagnant  water,  but  it  was  not  until  a  few  years  ago  that 
the  accuracy  of  this  assumption  was  actually  demonstrated. 
By  a  special  method  of  staining  Loffler1  rendered  visible 
these  hair-like  appendages.  His  method,  as  well  the  several 


FIG.  7 


a,  spiral  forms  with  a  flagellum  at  only  one  end;  6,  bacillus  of  typhoid 
fever  with  flagella  given  off  from  all  sides;  c,  large  spirals  from  stagnant 
water  with  wisps  of  flagella  at  their  ends  (spirillum  undula). 

modifications  that  have  been  made  of  it,  depends  for  success 
upon  the  use  of  mordants,  through  the  agency  of  which 
the  stains  employed  are  caused  to  adhere  with  increased 
tenacity  to  the  objects  under  treatment. 


1  Loffler's  method  of  staining  will  be  found  in   the  chapter  devoted  to 
this  part  of  the  technique.        v 


CHAPTER  III. 

Principles  of  Sterilization  by  Heat — Methods  Employed — Discontinued 
Sterilization — Fractional  Sterilization — Apparatus  Employed — Sterili- 
zation under  Pressure — Sterilization  by  Hot  Air — Thermal  Death- 
point  of  Bacteria — Chemical  Disinfection  and  Sterilization — Mode 
of  Action  of  Disinfectants — Practical  Disinfection. 

OF  fundamental  importance  to  successful  bacteriological 
manipulations  are  acquaintance  with  the  principles  under- 
lying the  methods  of  sterilization  and  disinfection,  and 
familiarity  with  the  approved  methods  of  applying  these 
principles  in  practice. 

In  many  laboratories  it  is  customary  to  employ  the  term 
sterilization  for  the  destruction  of  bacteria  by  heat,  and  the 
term  disinfection  for  the  accomplishment  of  the  same  end 
through  the  use  of  chemical  agents.  Such  distinction  in  the 
use  of  the  terms  is  obviously  incorrect,  as  we  shall  endeavor 
to  explain. 

The  laboratory  application  of  the  word  sterilization  for 
the  destruction  of  bacteria  by  high  temperatures  probably 
arose  from  the  circumstance  that  culture-media,  and  certain 
other  articles  that  it  is  desirable  to  render  free  from  bacterial 
life,  are  not  treated  by  chemical  agents  for  this  purpose, 
but  are  exposed  to  the  influence  of  heat  in  various  forms  of 
apparatus  known  as  sterilizers;  and  the  process  is,  therefore, 
known  as  sterilization.  On  the  other  hand,  cultures  no 
longer  useful,  bits  of  infected  tissue,  and  apparatus  generally 
that  it  is  desirable  to  render  free  from  danger,  are  com- 
monly subjected  for  a  time  to  the  action  of  chemical  com- 
pounds possessing  germicidal  properties — i.  e.,  to  the  action 
(76) 


PRINCIPLES  OF  STERILIZATION  77 

of  disinfectants;  and  the  process  is,  consequently,  known  as 
disinfection,  though  the  same  end  can  also  be  reached  by 
the  application  of  heat  to  these  articles.  Strictly  speaking, 
sterilization  implies  the  complete  destruction  of  the  vitality 
of  all  microorganisms  that  may  be  present  in  or  upon  the 
substance  to  be  sterilized,  and  can  be  accomplished  by  the 
proper  application  of  both  thermal  and  chemical  agents; 
while  disinfection,  though  it  may  insure  the  destruction  of 
all  living  forms  that  are  present,  need  not  of  necessity  do 
so,  but  may  be  limited  in  its  action  to  those  only  that  possess 
the  power  of  infecting;  it  may  or  may  not,  therefore,  be 
complete  in  the  sense  of  sterilization.  -From  this  we  see  it 
is  possible  to  accomplish  both  sterilization  and  disinfection 
as  well  by  chemical  as  by  thermal  means. 

In  practice  the  employment  of  these  means  is  governed 
by  circumstances.  In  the  laboratory  it  is  essential  that 
all  culture  media  with  which  work  is  to  be  conducted  should 
be  free  from  living  bacteria  or  their  spores — they  must  be 
sterile;  and  it  is  equally  important  that  their  original 
chemical  composition  should  remain  unchanged.  It  is 
evident,  therefore,  that  sterilization  of  these  substances 
by  means  of  chemicals  is  out  of  the  question,  for,  while  the 
media  could  be  thus  sterilized,  it  would  be  necessary,  in 
order  to  accomplish  this,  to  add  to  them  substances  cap- 
able not  only  of  destroying  all  microorganisms  present,  but 
whose  presence  would  at  the  same  time  prevent  the  growth 
of  bacteria  that  are  to  be  subsequently  cultivated  in  these 
media — that  is  to  say,  after  performing  their  sterilizing 
or  germicidal  function  the  chemical  disinfectants  would, 
by  their  further  presence,  exhibit  their  antiseptic  properties 
and  thus  render  the  material  useless  as  a  culture  medium. 
Exceptions  to  this  are  seen,  however,  in  the  case  of  certain 


78  BACTERIOLOGY 

volatile  substances  possessing  disinfectant  powers — chloro- 
form and  ether,  for  instance;  these  bodies,  after  exhibiting 
their  germicidal  activities,  may  be  driven  off  by  gentle  heat, 
leaving  the  media  quite  suitable  for  purposes  of  cultivation. 
They  are  not,  however,  in  general  use  in  this  capacity. 

The  circumstances  under  which  chemical  sterilization  or 
disinfection  is  practised  in  the  laboratory  are,  ordinarily, 
either  those  in  which  it  is  desirable  to  render  materials  free 
from  danger  that  are  not  affected  by  the  chemical  action 
of  the  agents  used,  such  as  glass  apparatus,  etc.,  or  where 
destructive  changes  in  the  composition  of  the  substances 
to  be  treated,  as  in-the  case  of  old  cultures,  infected  tissues, 
pathological  exudates,  feces,  etc.,  are  a  matter  of  no  conse- 
quence. On  the  other  hand,  for  the  sterilization  of  all 
materials  to  be  used  as  culture  media  heat  only  is  employed.1 

The  two  processes  will  be  explained  in  this  chapter, 
beginning  with 

STERILIZATION   BY   HEAT. 

Sterilization  by  means  of  high  temperature  is  accom- 
plished in  several  ways,  viz.,  by  subjecting  the  articles  to 
be  treated  to  a  high  temperature  in  a  properly  constructed 
oven — this  is  known  as  dry  sterilization;  by  subjecting 
them  to  the  action  of  streaming  or  live  steam  at  the  tem- 
perature of  100°  C.;  and  by  subjecting  them  to  the  action 
of  steam  under  pressure,  under  which  circumstance  the 
temperature  to  which  they  are  exposed  becomes  more  and 
more  elevated  as  the  pressure  increases. 

Experience  has  taught  us  that  the  process  of  sterilization 
by  dry  heat  is  of  limited  application  because  of  its  many 

1  An  occasional  exception  to  this  is  the  use  of  chloroform,  mentioned  above. 


STERILIZATION  BY  HEAT  79 

disadvantages.  For  successful  sterilization  by  the  method 
of  dry  heat,  not  only  is  a  relatively  high  temperature  needed, 
but  the  substances  under  treatment  must  be  exposed  to  this 
temperature  for  a  comparatively  long  time.  The  penetra- 
tion of  dry  heat  into  materials  which  are  to  be  sterilized  is, 
moreover,  much  less  thorough  than  that  of  steam.  Many 
substances  of  vegetable  and  animal  origin  are  rendered 
valueless  by  subjection  to  the  dry  method  of  sterilization. 
For  these  reasons  comparatively  few  materials  can  be 
sterilized  in  this  way  without  seriously  impairing  their 
further  usefulness. 

Successful  sterilization  by  dry  heat  cannot  usually  be 
accomplished  at  a  temperature  lower  than  150°  C.,  and  to 
this  degree  of  heat  the  objects  should  be  subjected  for  not 
less  than  one  hour.  For  the  sterilization,  therefore,  of  the 
organic  materials  of  which  the  media  employed  in  bacterio- 
logical work  are  composed,  and  of  domestic  articles,  such 
as  cotton,  woollen,  wooden,  and  leather  articles,  this  method 
is  wholly  unsuitable.  In  bacteriological  work  its  application 
is  limited  to  the  sterilization  of  glassware  principally — such, 
for  example,  as  flasks,  plates,  small  dishes,  test-tubes, 
pipettes — and  such  metal  instruments  as  are  not  seriously 
injured  by  the  high  temperature. 

Methods  Employed. — Sterilization  by  moist  heat — steam — 
offers  conditions  much  more  favorable.  The  penetrating 
power  of  the  steam  is  not  only  more  energetic,  but  the  tem- 
perature at  which  sterilization  is  ordinarily  accomplished  is, 
as  a  rule,  not  destructive  to  the  objects  under  treatment. 
This  is  conspicuously  seen  in  the  work  of  the  laboratory; 
the  culture  media,  composed  in  the  main  of  decomposable 
organic  materials  that  would  be  rendered  entirely  worthless 
if  exposed  to  the  dry  method  of  sterilization,  sustain  no 


80  BACTERIOLOGY 

injury  whatever  when  intelligently  subjected  to  an  equally 
effective  sterilization  with  steam.  The  same  may  be  said 
of  cotton  and  woollen  fabrics,  bedding,  clothing,  etc. 
•  Aside  from  the  relations  of  the  two  methods  to  the  mate- 
rials to  be  sterilized,  their  action  toward  the  organisms  to 
be  destroyed  is  quite  different.  The  penetrating  power  of 
steam  renders  it  by  far  the  more  efficient  agent  of  the  two. 
The  spores  of  several  organisms  which  are  killed  by  an 
exposure  of  but  a  few  moments  to  the  action  of  steam,  resist 
the  destructive  action  of  dry  heat  at  a  higher  temperature 
for  a  much  greater  length  of  time. 

These  differences  will  be  strikingly  brought  out  in  the 
experimental  work  on  this  subject.  For  our  purposes  it 
is  necessary  to  remember  that  the  two  methods  have  the 
following  applications : 

The  dry  method,  at  a  temperature  of  150°-180°  C.  for 
one  hour,  is  employed  for  the  sterilization  of  glassware  such 
as  flasks,  test-tubes,  culture-dishes,  pipettes,  plates,  etc. 

Sterilization  by  steam  is  practised  with  all  culture  media, 
whether'  fluid  or  solid.  Bouillon,  milk,  gelatin,  agar-agar, 
potato,  etc.,  are  under  no  circumstances  to  be  subjected  to 
dry  heat. 

Discontinued  Sterilization. — The  manner  in  which  heat  is 
employed  in  processes  of  sterilization  varies  with  circum- 
stances. When  used  in  the  dry  form  its  application  is  always 
continuous — i.  e.,  the  objects  to  be  sterilized  are  simply 
exposed  to  the  proper  temperature  for  the  length  of  time 
necessary  to  destroy  all  living  organisms  which  may  be  upon 
them.  With  the  use  of  steam,  on  the  other  hand,  the  articles 
to  be  sterilized  are  frequently  of  such  a  nature  that  a  pro- 
longed application  of  heat  might  materially  injure  them. 
For  this  and  other  reasons  steam  is  usually  applied  inter- 


STERILIZATION  BY  HEAT  81 

mittently  and  for  short  periods  of  time.  The  principles 
involved  in  the  intermittent  method  of  sterilization  depend 
upon  differences  of  resistance  to  heat  which  the  organisms 
to  be  destroyed  are  known  to  possess  at  different  stages 
of  their  development.  During  the  life  cycle  of  many  of  the 
bacilli  there  is  a  stage  in  which  the  resistance  of  the  organism 
to  the  action  of  both  chemical  and  thermal  agents  is  much 
greater  than  at  other  stages  of  their  development.  This 
increased  power  of  resistance  appears  when  these  organisms 
are  in  the  spore-  or  resting-stage,  to  which  reference  has 
already  been  made.  When  in  the  vegetative  or  growing 
stage  most  bacteria  are  killed  in  a  short  time  by  a  relatively 
low  temperature;  whereas,  under  conditions  which  favor  the 
production  of  spores,  the  spores  are  seen  to  be  capable  of 
resisting  very  much  higher  temperatures  for  an  appreciably 
longer  time;  indeed,  spores  of  certain  bacilli  have  been 
encountered  that  retain  the  power  of  germinating  after  an 
exposure  of  from  five  to  six  hours  to  the  temperature  of 
boiling  water.  Such  powers  of  resistance  have  never  been 
observed  in  the  vegetative  stage  of  development.  These 
differences  in  resistance  to  heat  which  the  spore-forming 
organisms  possess  at  their  different  stages  of  development  is 
taken  advantage  of  in  the  process  of  sterilization  by  steam 
known  as  the  discontinuous,  fractional,  or  intermittent 
method,  and  are  the  essential  feature  of  the  principles  on 
which  the  method  is  based. 

As  culture  media  are  dependent  for  their  usefulness  upon 
the  presence  of  more  or  less  unstable  organic  compounds, 
the  object  aimed  at  in  this  method  is  to  destroy  the  organ- 
isms in  the  shortest  time  and  with  the  least  amount  of  heat. 
It  is  accomplished  by  subjecting  them  to  the  elevated 
temperature  at  a  time  when  the  bacteria  are  in  the  vegetat- 
6 


82  BACTERIOLOGY 

ing  or  growing  stage — i.  e.,  the  stage  at  which  they  are  most 
susceptible  to  detrimental  influences.  In  order  to  accom- 
plish this  it  is  necessary  that  there  should  exist  conditions 
of  temperature,  nutrition,  and  moisture  which  favor  the 
vegetation  of  the  bacilli  and  the  germination  of  any  spores 
that  may  be  present.  When,  as  in  freshly  prepared  nutrient 
media,  this  combination  is  found,  the  spore-forming  organ- 
isms are  not  only  less  likely  to  enter  the  spore-stage  than 
when  their  environment  is  less  favorable  to  their  vegetation, 
but  spores  which  may  already  exist  develop  very  quickly 
into  mature  cells. 

It  is  plain,  then,  that  with  the  first  application  of  steam 
to  the  substance  to  be  sterilized  the  mature  vegetative  forms 
are  destroyed;  while  certain  spores  that  may  be  present 
resist  this  treatment,  providing  the  sterilization  is  not  con- 
tinued for  too  long  a  time.  If  now  the  sterilization  be 
discontinued,  and  the  material  which  presents  conditions 
favorable  to  the  germination  of  the  spores  be  allowed  to 
stand  for  a  time,  usually  for  about  twenty-four  hours,  at 
a  temperature  of  from  20°  to  22°  C.,  those  spores  which 
resisted  the  action  of  the  steam  will,  in  the  course  of  this 
interval,  germinate  into  the  less  resistant  vegetative  cells. 
A  second  short  exposure  to  the  steam  kills  these  forms  in 
turn,  and  by  a  repetition  of  this  process  all  bacteria  that 
were  present  may  be  destroyed  without  the  application  of 
the  steam  having  been  of  long  duration  at  any  time.  It 
should  be  remembered  that  while  spores  which  may  be 
present  are  not  directly  killed  by  such  an  exposure  to  heat 
as  they  experience  in  the  intermittent  method  of  sterili- 
zation, still  their  power  of  germination  is  somewhat  inhibited 
by  this  treatment.  In  this  method,  therefore,  if  the  tem- 
perature of  100°  C.  be  employed  for  too  long  a  time,  it  is 


STERILIZATION  BY  HEAT  83 

possible  so  to  retard  the  germination  of  the  spores  as  to 
render  it  impossible  for  them  to  develop  into  the  vegetative 
stage  during  the  interval  between  the  heatings.  By  exces- 
sively long  exposures  to  high  temperature,  but  not  long 
enough  to  destroy  the  spores  directly,  the  object  aimed  at 
in  the  method  may  be  defeated,  and  in  the  end  the  substance 
undergoing  sterilization  be  found  still  to  contain  living 
bacteria.  In  this  process  the  plan  that  has  given  most  satis- 
factory results  is  to  subject  the  materials  to  be  sterilized 
to  the  action  of  steam,  under  the  ordinary  conditions  of 
atmospheric  pressure,  for  fifteen  minutes  on  each  of  three 
successive  days,  and  during  the  intervals  to  maintain  them 
at  a  temperature  of  about  25°-30°  C.  At  the  end  of  this 
time  all  living  organisms  which  were  present  will,  as  a  general 
rule,  have  been  destroyed,  and,  unless  opportunity  is  given 
for  the  access  of  new  organisms  from  without,  the  substances 
thus  treated  remain  sterile.  As  an  exception  to  this,  certain 
species  of  spore-forming  bacteria  are  occasionally  encountered 
that  are  not  readily  destroyed  by  this  mode  of  treatment. 
These  species  are  found  so  uniformly  in  the  soil  that  the 
customary  designation  for  them  is  that  of  "the  soil  bacteria." 
This  group  includes  a  number  of  species  that  are  endowed 
with  remarkable  resistance  to  heat.  Some  of  them  are 
probably  thermophilic  by  nature,  which  would  account  not 
only  for  the  failure  to  destroy  their  spores  by  the  ordinary 
exposures  to  steam,  but  also  for  their  slow  and  incomplete 
development  from  the  spore  to  the  less  resistant  vegetative 
stage  during  the  intervals  between  the  heatings,  for,  as  a 
rule,  the  materials  containing  them  are  kept  at  a  temperature 
during  these  intervals  that  is  too  low  to  favor  the  rapid 
germination  of  the  species  having  thermophilic  tendencies. 
As  a  result  of  the  presence  of  these  species,  media  that 


84  BACTERIOLOGY 

have  been  subjected  to  the  customary  discontinuous  method 
of  sterilization  may,  after  having  been  kept  for  a  time, 
reveal  the  presence  of  isolated  colonies  of  bacteria  distrib- 
uted through  them  in  such  a  way  as  to  preclude  all  likelihood 
of  their  having  fallen  upon  it  from  the  air  after  sterilization 
was  supposedly  complete. 

Theobald  Smith1  has  called  attention  to  an  instructive 
personal  experience.  He  finds  that  when  media  are  present 
in  vessels  in  only  thin  layers  the  spores  of  anaerobic  specie's 
do  not  develop  into  the  vegetative  forms  during  the  interval 
between  the  heatings,  for  the  reason  that  the  shallow  layer 
of  medium  does  not  sufficiently  exclude  free  oxygen  to  per- 
mit it;  and  by  subjecting  such  materials,  apparently  steril- 
ized by  the  intermittent  method,  to  strictly  anaerobic 
conditions  a  development  of  anaerobic  species  will  often 
occur.  On  the  other  hand,  if  the  vessels  be  nearly  filled  with 
media,  and  especially  if  the  area  of  the  surface  be  small,  the 
conditions  are  much  more  favorable  to  the  germination  of 
anaerobic  spores,  and  sterilization  by  this  process  after  such 
precautions  is  usually  perfect. 

Fortunately,  these  undesirable  experiences  are  rare,  but 
that  they  do  occur,  and  result  in  no  small  degree  of  annoy- 
ance, will  be  admitted  by  most  bacteriologists. 

It  must  be  borne  in  mind  that  this  method  of  sterilization 
is  only  applicable  in  those  cases  which  present  conditions 
favorable  to  the  germination  of  the  spores  into  mature 
vegetative  cells.  Dry  substances,  such  as  instruments, 
bandages,  apparatus,  etc.,  or  organic  materials  in  which 
decomposition  is  far  advanced,  where  conditions  of  nutrition 
favorable  to  the  germination  of  spores  are  not  present,  do 
not  offer  the  conditions  requisite  for  the  successful  operation 

1  Journal  of  Experimental  Medicine,  iii,  No.  6,  p.  647. 


STERILIZATION  BY  HEAT  85 

of  the  principles  underlying  the  intermittent  method  of 
sterilization. 

Discontinued  Sterilization  at  Low  Temperatures. — The  pro- 
cess of  discontinued  sterilization  at  low  temperatures  is 
based  upon  exactly  the  same  principle,  but  differs  in  two 
respects  from  the  foregoing  in  the  manner  by  which  it  is 
practised,  viz.,  it  requires  a  greater  number  of  exposures 
for  its  accomplishment,  and  the  temperature  at  which  it 
is  conducted  is  not  above  68°-70°  C.  It  is  employed  for 
the  sterilization  of  easily  decomposable  materials,  which 
would  be  rendered  useless  by  steam,  but  which  are  unal- 
tered by  the  temperature  employed,  and  for  certain  albu- 
minous culture  media  that  it  is  desirable  to  retain  in  a 
fluid  condition  during  sterilization,  but  which  would  be 
coagulated  if  exposed  to  higher  temperatures.  This  pro- 
cess requires  that  the  material  to  be  sterilized  should  be 
subjected  to  a  temperature  of  68°-70°  C.  for  one  hour  on 
each  of  six  successive  days,  the  interval  of  twenty-four 
hours  between  the  exposures  admitting  of  the  germination 
of  spores  into  mature  cells.  During  this  interval  the  sub- 
stances under  treatment  are  kept  at  about  25°-30°  C.  The 
temperature  employed  in  this  process  suffices  to  destroy, 
in  about  one  hour,  the  vitality  of  almost  all  organisms  in 
the  vegetative  stage.  Formerly  blood  serum  was  always 
sterilized  by  the  intermittent  method  at  a  low  temperature. 

Direct  Sterilization. — Sterilization  by  steam  is  also  prac- 
tised by  what  may  be  called  the  direct  method — that  is  to 
say,  both  the  mature  organisms  and  the  spores  which  may 
be  present  in  the  material  to  be  sterilized  are  destroyed 
by  a  single  exposure  to  the  steam.  In  this  method  steam 
at  its  ordinary  temperature  and  pressure — live  steam  or 
streaming  steam,  as  it  is  called — is  employed  just  as  in  the 


86  BACTERIOLOGY 

first  method  described;  but  it  is  allowed  to  act  for  a  much 
longer  time,  usually  for  not  less  than  an  hour;  or  steam  under 
pressure,  and  consequently  of  a  higher  temperature,  is  now 
frequently  employed.  By  the  latter  procedure  a  single 
exposure  of  fifteen  minutes  is  sufficient  for  the  destruction 
of  practically  all  bacilli  and  their  spores,  providing  the 
pressure  of  the  steam  is  not  less  than  one  atmosphere  over 
and  above  that  of  normal;  this  is  approximately  equivalent 
to  a  temperature  of  122°  C.  to  which  the  organisms  are 
exposed. 

The  objection  that  has  been  urged  to  both  of  these 
methods,  particularly  that  in  which  steam  under  pressure 
is  employed,  is  that  the  properties  of  the  media  are  altered. 
Gelatin  is  said  to  become  cloudy  and  lose  the  property  of 
solidifying;  in  bouillon  and  agar-agar  fine  precipitates  are 
said  to  result,  and  some  believe  the  reaction  undergoes  a 
change.  In  the  experience  of  those  who  have  used  steam 
under  pressure  not  exceeding  one  atmosphere  for  ten  to 
fifteen  minutes  these  obstacles  have  rarely  been  encoun- 
tered. There  is  one  point  to  be  borne  in  mind,  however,  in 
using  steam  under  pressure,  viz.,  it  is  not  possible  to  regulate 
the  time  of  exposure  to  the  same  degree  of  nicety  as  where 
ordinary  live  steam  is  used.  The  reason  for  this  is  that  if 
the  apparatus  be  opened  to  remove  the  objects  being  steril- 
ized while  the  steam  within  it  is  under  pressure,  the  escape 
of  steam  will  be  so  rapid  that  all  fluids  within  the  chamber, 
thus  suddenly  relieved  of  pressure,  will  begin  to  boil  violently, 
and,  as  a  rule,  will  boil  quite  out  of  the  tubes,  flasks,  etc., 
containing  them.  For  this  reason  the  apparatus  must  be 
kept  closed  until  cool,  or  until  the  gauge  indicates  that 
pressure  no  longer  exists  within  the  chamber,  and  even 
then  it  should  be  opened  very  cautiously.  It  is  patent  from 


STERILIZATION  BY  HEAT  87 

this  that  the  temperature  and  time  of  exposure  of  articles 
sterilized  by  this  process  cannot  usually  be  controlled  with 
accuracy.  It  requires  some  time  to  reach  a  given  pressure 
after  the  apparatus  is  closed,  and  it  also  requires  time  for 
cooling  after  the  desired  exposure  to  such  pressure  before 
the  apparatus  can  be  opened. 

It  is  manifest  that  during  these  three  periods,  viz.,  (a) 
reaching  the  pressure  desired,  (6)  time  during  which  the 
pressure  is  maintained,  and  (c)  time  for  fall  of  pressure 
before  the  chamber  can  be  opened,  it  is  difficult  to  say 
certainly  to  what  temperature  and  pressure  the  articles  in 
the  apparatus  have,  on  the  whole,  been  subjected.  Clearly, 
if  the  desired  pressure  and  temperature  have  been  maintained 
for  ten  minutes,  one  cannot  say  that  that  is  all  the  heat  to 
which  the  articles  have  been  subjected  during  their  stay 
in  the  chamber.  In  this  light,  while  steam  under  pressure 
may  answer  very  well  for  routine  sterilization,  still  it  pre- 
sents insurmountable  obstacles  to  its  use  in  more  delicate 
experiments  where  time-exposure  to  definite  temperature  is 
of  importance.  Nevertheless,  for  general  laboratory  pur- 
poses, sterilization  by  steam  under  pressure  has  so  much 
to  recommend  it  in  the  way  of  economy  of  time  and  cerr 
tainty  of  accomplishment  that  it  has  practically  superseded 
the  older  methods  of  sterilization  by  streaming  or  live  steam; 
and  in  most  laboratories  the  original  styles  of  steam  steril- 
izers are  rapidly  giving  way  to  some  one  or  another  of  the 
modern  forms  of  autoclave. 

For  sterilization  by  live  steam  the  apparatus  in  common 
use  was  for  a  long  time  the  cylindrical  boiler  recommended 
by  Koch.  (See  Fig.  8.)  Its  construction  is  very  simple, 
essentially  that  of  the  ordinary  domestic  potato-steamer. 
It  consists  of  a  copper  cylinder,  the  lower  fifth,  approximately, 


88  BACTERIOLOGY 

of  which  is  somewhat  larger  in  circumference  than  the 
remaining  four-fifths  and  serves  as  a  reservoir  for  the  water 
from  which  the  steam  is  to  be  generated.  Covering  this 
section  of  the  cylinder  is  a  wire  rack  or  grating,  through 
which  the  steam  passes,  and  which  supports  the  articles  to 
be  sterilized.  Above  this,  comprising  the  remaining  four- 

FIG.  8 


Steam  sterilizer,  pattern  of  Koch. 

fifths  of  the  cylinder,  is  the  chamber  for  the  reception  of 
the  materials  over  and  through  which  the  steam  is  to  pass. 
The  cylinder  is  closed  by  a  snugly  fitting  cover,  through 
which  are  usually  two  perforations,  into  which  a  thermo- 
meter and  a  manometer  may  be  inserted.  The  whole 
of  the  outer  surface  of  the  apparatus  is  encased  in  a  non- 
conducting mantle  of  asbestos  or  felt. 


STERILIZATION  BY  HEAT  89 

The  water  is  heated  by  a  gas-flame  placed  in  an  enclosed 
chamber,  upon  which  the  apparatus  rests,  which  serves  to 
diminish  the  loss  of  heat  and  deflection  of  the  flame  through 
the  action  of  draughts.  The  apparatus  is  simple  in  con- 
struction, and  the  only  point  which  is  to  be  observed  while 
using  it  is  the  level  of  the  water  in  the  reservoir.  On  the 
reservoir  is  a  water-gauge  which  indicates  at  all  times  the 


FIG.  9 


Arnold  steam  sterilizer. 

amount  of  water  in  the  apparatus.  The  amount  of  water 
should  never  be  too  small  to  be  indicated  by  the  gauge; 
otherwise  there  is  danger  of  the  reservoir  becoming  dry  and 
the  bottom  of  the  apparatus  being  destroyed  by  the  direct 
action  of  the  flame. 

A  sterilizer  that  has  come  into  very  general  use  in  bac- 
teriological laboratories  is  one  originally  intended  for  use 


90  BACTERIOLOGY 

in  the  kitchen.  It  is  called  the  "Arnold  steam  sterilizer." 
It  is  very  ingenious  in  its  construction  as  well  as  economical 
in  its  employment. 

The  difference  between  this  apparatus  and  that  just 
described  is  that  it  provides  for  the  condensation  of  the 
steam  after  its  escape  from  the  sterilizing  chamber,  and 
returns  the  water  of  condensation  automatically  to  the 
reservoir,  so  that  in  practice  the  apparatus  requires  but 
little  attention,  as  with  ordinary  care  there  is  no  likelihood 
of  the  water  in  the  reservoir  becoming  exhausted,  with  the 
consequent  destruction  of  the  sterilizer.  Fig.  9  shows  a 
section  through  this  apparatus.  » 

STERILIZATION   UNDER  PRESSURE. 

The  advantages  of  the  use  of  steam  under  pressure  for 
the  purposes  of  sterilization  have  received  such  general 
recognition  that  almost  everywhere  this  method  is  sup- 
planting the  older  one  of  intermittent  sterilization  with 
streaming  or  live  steam.  By  this  plan  one  is  able  to  accom- 
plish, by  a  single  exposure  of  fifteen  minutes  to  steam  under 
a  pressure  of  one  atmosphere,  the  same  end  that  would, 
with  streaming  steam,  require  three  exposures  of  fifteen 
minutes  on  each  of  three  successive  days. 

For  sterilization  by  steam  under  pressure  several  special 
forms  of  apparatus  exist.  The  principles  involved  in  them 
all  are,  however,  the  same.  They  provide  for  the  generation 
of  steam  in  a  chamber  from  which  it  cannot  escape  when 
the  apparatus  is  closed.  Upon  the  cover  of  this  chamber 
is  a  safety-valve,  which  can  be  regulated  so  that  any  degree 
of  pressure  (and  coincidently  of  temperature)  that  is  desir- 
able may  be  maintained  within  the  sterilizing  chamber. 


STERILIZATION  BY  HOT  AIR 


91 


These  sterilizers  are  known  as  "digesters"  and  as  "auto- 
claves." Their  construction  can  best  be  understood  by 
reference  to  Figs.  10  and  11. 


FIG.  10 


A  B 

Autoclave.    A,  external  appearance;  B,  section. 


STERILIZATION   BY   HOT   AIR. 

The  hot-air  sterilizers  used  in  laboratories  are  simply 
double-walled  boxes  of  Russian  or  Swedish  iron  (Fig.  12), 
having  a  double-walled  door,  which  closes  tightly,  and  a 
heavy  copper  bottom.  They  are  provided  with  openings 
for  the  escape  of  the  contained  air  and  the  entrance  of  the 
heated  air.  The  flame,  usually  from  a  rose-burner  (Fig.  13), 


92  BACTERIOLOGY 

is  applied  directly  to  the  bottom.  The  heat  circulates  from 
the  lower  surface  around  about  the  apparatus  through  the 
space  between  its  walls. 

FIG.  11 


Autoclave  or  digester  for  sterilizing  by  steam  under  pressure. 

The  construction  of  the  copper  bottom  of  the  apparatus 
upon  which  the  flame  impinges  is  designed  to  prevent  the 
direct  action  of  the  flame  upon  the  sheet-iron  bottom  of  the 
chamber.  It  consists  of  several  copper  plates  placed  one 


STERILIZATION  BY  HOT  AIR 


93 


above  the  other,  but  with  a  space  of  about  4  to  5  mm. 
between  the  plates.  These  copper  bottoms  after  a  time 
become  burned  out,  and  unless  they  are  replaced  the  appara- 
tus is  useless.  The  older  forms  of  hot-air  sterilizers  are  so 
constructed  that  their  repair  is  a  matter  involving  some  time 
and  expense.  To  meet  this  objection  I  had  constructed 


FIG.  12 


FIG.  13 


Laboratory  hot-air  sterilizer. 


Rose-burner. 


some  years  ago  a  sterilizer  in  all  respects  similar  to  the  old 
form  except  in  the  arrangement  of  the  copper  bottom.  This 
latter  is  made  in  such  a  way  that  it  can  easily  be  removed, 
so  that  by  keeping  several  sets  of  copper  plates  on  hand 
a  new  plate  can  readily  be  inserted  when  the  old  one  is 
burned  out. 

In  the  employment  of  the  hot-air  sterilizer  care  should 


94  BACTERIOLOGY 

always  be  given  to  the  condition  of  the  copper  bottom;  for 
the  direct  application  of  heat  to  the  sheet-iron  plate  upon 
which  the  substances  to  be  sterilized  stand  results  not  only 
in  destruction  of  the  apparatus,  but  frequently  in  destruc- 
tion of  the  substances  undergoing  sterilization. 

Since  the  temperature  at  which  this  form  of  sterilization 
is  usually  accomplished  is  high,  from  150°  to  180°  C.,  it  is 
well  to  have  the  apparatus  encased  in  asbestos  boards,  to 
diminish  the  radiation  of  heat  from  its  surfaces.  This  not 
only  confines  the  heat  to  the  apparatus,  but  guards  against 
the  destructive  action  of  the  radiated  heat  on  woodwork, 
furniture,  etc.,  that  may  be  in  the  neighborhood. 

Thermal  Death-point  of  Bacteria. — By  "thermal  death- 
point  of  bacteria"  is  meant  the  temperature  necessary  to 
kill  them  in  a  given  time.  As  this  varies  with  different 
species,  it  is  an  aid  to  identification.  For  the  practical  pur- 
poses of  the  sanitarian  the  knowledge  is  of  fundamental 
importance.  The  thermal  death-point  of  an  organism  is 
ascertained  by  subjecting  it  to  varying  degrees  of  tempera- 
ture for  five  or  ten  minutes  until  the  point  is  reached  where 
it  is  killed.  The  test  is  best  carried  out  by  means  of  small 
glass  bulbs,  the  so-called  Sternberg  bulbs,  or  through  the 
use  of  capillary  tubes  containing  a  small  amount  of  fluid 
inoculated  with  the  organism  to  be  studied.  The  bulb, 
or  tube,  is  sealed  in  the  gas  flame  and  placed  in  a  water- 
bath  kept  at  50°  C.  for  five  minutes.  Sub-cultures  are  now 
made  to  learn  whether  the  bacteria  have  been  killed  or  not. 
If  the  organism  survives  the  test  is  repeated  at  55°,  60°,  65  °, 
and  70°  C.  Finally,  the  test  is  repeated  for  each  degree  of 
temperature  between  the  points  where  growth  is  still  apparent 
and  where  the  organisms  have  been  killed.  If  the  bacteria 
were  killed  when  heated  to  60°  C.  for  five  minutes,  but  sur- 


CHEMICAL  STERILIZATION  AND  DISINFECTION     95 

vived  when  heated  to  55°  C.,  then  similar  tests  are  made  for 
the  same  length  of  time  for  each  degree  of  temperature 
between  55°  and  60°  C.  It  will  usually  be  found  that  heating 
for  ten  minutes  suffices  to  kill  the  bacteria  at  a  temperature 
one  or  two  degrees  lower  than  that  required  when  heated 
for  only  five  minutes.  All  such  tests  should  be  made  at 
least  in  duplicate,  and  the  mean  of  the  results  taken. 

CHEMICAL   STERILIZATION    AND   DISINFECTION. 

As  has  been  stated,  it  is  possible  by  means  of  certain 
chemical  substances  to  destroy  all  bacteria  and  their  spores 
that  may  be  within  or  upon  various  materials  and  objects — 
i.  e.,  to  sterilize  them;  and  it  is  also  possible  by  the  same 
means  to  rob  objects  of  their  dangerous  infective  properties 
without  at  the  same  time  sterilizing  them — i.  e.,  to  disinfect 
them.  This  latter  process  depends  upon  the  fact  that  the 
vitality  of  many  of  the  less  resistant  pathogenic  organisms 
is  easily  destroyed  by  an  exposure  to  particular  chemical 
substances  that  may  be  without  effect  upon  the  more  resis- 
tant saprophytes  and  their  spores  that  are  present. 

In  general,  the  use  of  chemicals  for  sterilization  is  not  to 
be  considered  in  connection  with  substances  that  are  to  be 
employed  as  culture  media,  and  their  employment  is  re- 
stricted in  the  laboratory  to  materials  that  are  of  no  further 
value,  and  to  infected  articles  that  are  not  injured  by  the 
action  of  the  agents  used,  though  exceptionally  such  vola- 
tile germicides  as  chloroform  and  ether  are  employed  for 
the  sterilization  of  special  culture-media.  (See  Preservation 
of  Blood-serum  with  Chloroform.)  In  short,  they  are  mainly 
of  value  in  rendering  infected  waste-material  innocuous. 
For  the  successful  performance  of  this  form  of  disinfection 


96  BACTERIOLOGY 

there  is  one  fundamental  rule  always  to  be  borne  in  mind, 
viz.,  it  is  essential  to  success  that  the  disinfectant  used 
should  come  in  direct  contact  with  the  bacteria  to  be  de- 
stroyed, otherwise  there  is  no  disinfection. 

For  this  reason  one  should  always  remember,  in  selecting 
the  disinfecting  agent,  the  nature  of  the  materials  containing 
the  bacteria  upon  which  it  is  to  act,  for  the  majority  of 
disinfectants,  and  particularly  those  of  an  inorganic  nature, 
vary  in  the  degree  of  their  potency  with  the  chemical  nature 
of  the  mass  to  which  they  are  applied.  Often  the  materials 
containing  the  bacteria  to  be  destroyed  are  of  such  a  character 
that  they  combine  with  the  disinfecting  agent  to  form  insol- 
uble, more  or  less  inert  precipitates;  these  so  interfere  with 
the  penetration  of  the  disinfectant  that  many  bacteria  may 
escape  its  destructive  action  entirely  and  no  disinfection 
be  accomplished,  although  an  agent  may  have  been  employed 
that  would,  under  other  circumstances,  have  given  entirely 
satisfactory  results. 

An  antiseptic  is  a  body  which,  by  its  presence,  prevents 
the  growth  of  bacteria  without  of  necessity  killing  them. 
A  body  may  be  an  antiseptic  without  possessing  disinfecting 
properties  to  any  very  high  degree,  but  a  disinfectant  is 
always  an  antiseptic  as  well. 

A  germicide  is  a  body  possessing  the  property  of  killing 
bacteria. 

Mode  of  Action  of  Disinfectants. — In  the  destruction  of 
bacteria  by  means  of  chemical  substances  there  occurs, 
most  probably,  a  definite  chemical  reaction— that  is  to 
say,  the  characteristics  both  of  the  bacteria  and  the  agent 
employed  in  their  destruction  are  lost  in  the  production  of 
an  inert  third  body,  the  result  of  their  combination.  It  is 
impossible  to  state  with  certainty,  as  yet,  that  this  is  in 


CHEMICAL  STERILIZATION  AND  DISINFECTION     97 

general  the  case;  but  the  evidence  that  is  rapidly  accruing 
from  studies  upon  disinfectants  and  their  mode  of  action 
points  strongly  to  the  accuracy  of  this  belief.  This  reaction, 
in  which  the  typical  structures  of  both  bodies  concerned  are 
lost,  takes  place  between  the  agent  employed  for  disinfection 
and  the  protoplasm  of  the  bacteria.  For  example,  in  the 
reaction  that  is  seen  to  take  place  between  the  salts  of  mer- 
cury and  albuminous  bodies  there  results  a  third  compound, 
which  has  neither  all  the  characteristics  of  mercury  nor  of 
albumin,  but  partakes  of  some  of  the  peculiarities  of  both; 
it  is  a  combination  of  albumin  and  mercury,  commonly 
known  by  the  indefinite  term  "albuminate  of  mercury." 
Some  such  reaction  as  this  apparently  occurs  when  the 
soluble  salts  of  mercury  are  brought  in  contact  with 
bacteria.  This  view  has  been  strengthened  by  the  experi- 
ments of  Geppert,  in  which  the  reaction  was  caused  to  take 
place  between  the  spores  of  the  anthrax  bacillus  and  a 
solution  of  mercuric  chloride,  the  result  being  the  apparent 
destruction  of  the  vitality  of  the  spores  by  the  formation  of 
this  third,  inert  compound.  In  these  experiments  it  was 
shown  that  though  this  combination  had  taken  place,  still 
it  did  not  of  necessity  imply  the  death  of  the  spores,  for  if 
by  proper  means  the  combination  of  mercury  with  their 
protoplasm  was  broken  up,  many  of  the  spores  resumed 
their  vitality,  with  all  their  previous  disease  producing  and 
cultural  peculiarities.  Geppert  employed  a  solution  of  am- 
monium sulphide  for  the  purpose  of  destroying  the  combi- 
nation of  spore  protoplasm  and  mercury;  the  mercury  was 
precipitated  from  the  protoplasm  as  an  insoluble  sulphide, 
and  the  protoplasm  of  the  spores  returned  to  its  original 
condition.  These  and  other  somewhat  similar  experiments 
have  given  a  new  impulse  to  the  study  of  disinfectants,  and 
7 


98  BACTERIOLOGY 

in  the  light  shed  by  them  many  of  our  previously  formed 
ideas  concerning  the  action  of  disinfecting  agents  have 
been  modified. 

The  process  of  disinfection  is  not  a  catalytic  one — 'i.  e., 
occurring  simply  as  xa  result  of  the  presence  of  the  disin- 
fecting body,  which  is  not  itself  decomposed  during  its 
process  of  destruction — but  is,  as  said,  a  definite  chemical 
reaction  occurring  within  more  or  less  fixed  limits;  that  is 
to  say,  with  a  given  amount  of  the  disinfectant  just  so  much 
work,  expressed  in  terms  of  destruction  of  bacteria  can  be 
accomplished. 

Another  point  in  favor  of  this  view  is  the  increased 
energy  of  the  reaction  with  elevation  of  temperature.  Just 
as  in  other  chemical  phenomena  the  intensity  and 
rapidity  of  the  reaction  become  greater  under  the  influence 
of  heat,  so  in  the  process  of  disinfection  the  combination 
between  the  disinfectant  and  the  organisms  to  be  destroyed 
is  much  more  energetic  at  a  temperature  of  37°-39°  C. 
than  it  is  at  12°-15°  C. 

A  number  of  important  and  novel  suggestions  with  regard 
to  the  modus  operandi  of  disinfection  were  brought  out 
through  the  work  of  Kronig  and  Paul,1  who  took  up  the 
subject  from  its  physico-chemical  standpoint.  The  compre- 
hensive nature  of  this  elaborate  investigation  precludes 
more  than  a  brief  mention  of  some  of  the  conclusions  reached, 
and  in  order  that  these  may  be  intelligible,  certain  beliefs 
(working  hypotheses)  of  the  physical  chemists  should  be 
borne  in  mind.  In  1887  Arrhenius  proposed  the  theory 
that  when  an  electrolyte  (a  compound  decomposable  by  an 
electric  current)  is  dissolved  in  water  its  molecules  break 
down,  not  simply  into  their  component  atoms,  but  into 

1  Zeitschrift  fur  Hygiene  und  Infektionskrankheiten,  1897,  xxv,  1-112. 


CHEMICAL  STERILIZATION  AND  DISINFECTION,  99 

ions,  which  are  atoms  or  groups  of  atoms  having  electro- 
positive and  electro-negative  characteristics.  According 
to  this  theory,  salts,  when  dissolved  in  water,  undergo 
electrolytic  dissociation  into  metallic  and  acidic  ions,  the 
former  being  the  electro-positive  cation,  the  latter  the 
electro-negative  anion;  sodium  chloride,  for  example,  re- 
solving itself,  under  these  conditions,  into  its  sodium, 
or  metal  ion,  and  its  chlorine,  or  acidic  ion.  The  electro- 
positive cations,  according  to  Ostwald,  comprise  the  metals 
and  metal-like  radicals,  such  as  ammonium  (NH4)  and  hydro- 
gen (H) ;  while  the  electro-negative  anions  include  the  halo- 
gens, the  acidic  radicals  (such  as  NO3  and  SO4),  and  hydrosyl.1 
Using  this  theory  as  the  basis  of  their  investigations,  Kronig 
and  Paul  reached  the  following  conclusions  with  regard  to 
the  action  of  chemical  disinfectants: 

The  germicidal  value  of  a  metallic  salt  depends  not  only 
upon  its  specific  character,  but  also  upon  that  of  its  anion. 

Solutions  of  metallic  salts  in  which  the  metallic  part  is 
represented  by  a  complex  ion  and  in  which  the  concentra- 
tion of  the  metal  ion  is  very  slight,  have  but  feeble  disin- 
fecting activity. 

The  halogen  compounds  of  mercury  act  according  to  the 
degree  of  their  dissociation. 

The  disinfecting  power  of  the  halogens — chlorine,  bromine, 
iodine — (as  well  as  their  compounds)  is  in  inverse  ratio  to 
their  atomic  weights. 

The  disinfecting  activity  of  watery  solutions  of  mercuric 
chloride  is  diminished  by  the  addition  to  them  of  other 

1  Consult  Ostwald's  Lehrbuch  der  Allg.  Chemie;  or  Muir's  transla- 
tion of  Ostwald's  Solutions,  p.  189,  published  by  Longmans,  Green  & 
Co.,  London  and  New  York,  1891.  Also  The  Rise  of  the  Theory  of  Elec- 
trolytic Dissociation,  etc.,  by  H.  C.  Jones,  Ph.D.,  Johns  Hopkins  Hospital 
Bulletin,  No.  87,  June,  1898,  p.  136. 


100  BACTERIOLOGY 

halogen  compounds  of  metals  and  of  hydrochloric  acid.  It 
appears  probable  that  this  is  due  to  obstruction  offered  to 
electrolytic  dissociation. 

The  disinfecting  activities  of  watery  solutions  of  mer- 
curic nitrate,  mercuric  sulphate,  and  mercuric  acetate  are 
increased  by  the  moderate  addition  of  sodium  chloride. 

In  general,  acids  disinfect  according  to  the  degree  of  their 
dissociation — i.  e.,  according  to  the  concentration  of  their 
hydrogen  ions  in  the  solution. 

The  bases,  potassium,  sodium,  lithium,  and  ammonium 
hydroxide,  disinfect  according  to  the  degree  of  their  dis- 
sociation— i.  e.,  corresponding  to  the  concentration  of  their 
hydroxyl  ions  in  the  solution. 

The  disinfecting  activity  of  metallic  salts  is,  in  general, 
less  in  albuminous  fluids  than  in  water.  It  is  probable  that 
this  is  due  to  a  diminution  in  the  concentration  of  metallic 
ions  in  the  solution. 

The  reaction  between  the  inorganic  salts  and  albuminous 
bodies  is  not  selective;  they  combine  in  most  instances  with 
any  or  all  protoplasmic  bodies  present.  For  this  reason 
the  employment  of  many  of  the  commoner  disinfectants 
in  general  practice  is  a  matter  of  doubtful  advantage.  For 
example,  the  disinfection  of  excreta,  sputum,  or  blood, 
containing  pathogenic  organisms,  by  means  of  corrosive 
sublimate,  is  a  procedure  of  questionable  success.  The 
amount  of  sublimate  employed  may  be  entirely  used  up 
and  rendered  inactive  as  a  disinfectant  by  the  ordinary 
protoplasmic  substances  present,  without  having  any 
appreciable  effect  upon  the  bacteria  which  may  be  in  the 
mass. 

These  remarks  are  introduced  in  order  to  guard  against 
the  implicit  confidence  so  often  placed  in  the  disinfecting 


CHEMICAL  STERILIZATION  AND  DISINFECTION     101 

value  of  corrosive  sublimate.  In  many  bacteriological 
laboratories  it  is  the  custom  to  keep  at  hand  vessels  con- 
taining solutions  of  corrosive  sublimate,  into  which  i;nfec- 
tious  materials  may  be  placed.  The  value  of  this  procedure, 
as  we  have  just  learned,  may  be  more  or  less  questionable, 
especially  in  those  cases  in  which  the  substance  to  be  disin- 
fected is  of  a  proteid  nature  and  where  the  solution  used  is 
not  freshly  prepared  and  frequently  replenished."  £)n  the 
introduction  of  such  substances  into  the  sublimate  solution 
the  mercury  is  quickly  precipitated  by  "the*  alSuravri,1 '.a toil* it* 
disinfecting  properties  may  be  in  large  part  or  entirely 
destroyed;  we  may  in  a  very  short  time  have  little  else 
than  water  containing  an  inactive  precipitate  of  albumin 
and  mercury,  in  so  far  as  its  value  as  a  disinfectant  is  con- 
cerned. 

Though  the  other  inorganic  salts  have  not  been  so 
thoroughly  studied  in  this  connection,  it  is  nevertheless 
probable  that  the  same  precautions  should  be  taken  in 
their  employment  as  we  now  know  to  be  necessary  in  the 
use  of  the  salts  of  mercury. 

The  modes  of  action  of  other  germicides  have  not  been  so 
carefully  investigated  as  has  that  of  the  metallic  salts. 
From  the  nature  of  many  of  them,  however,  we  may  infer 
that  some  act  through  oxidation,  as  in  the  case  of  strong 
acids  and  other  active  oxidizers;  others  by  coagulation  or 
by  dehydration,  as  in  the  case  of  strong  aldehydes  and 
alcohols;  and  others  by  penetrating  the  cell  wall  and  fatally 
poisoning  the  bacterial  protoplasm,  as  in  the  case  of  hydro- 
cyanic acid  and  its  compounds. 

Practical  Disinfection. — Where,  it  is  desirable  to  use  chemi- 
cal disinfectants  in  the  laboratory,  much  more  satisfactory 
results  can  usually  be  obtained  from  the  employment  of 


102  BACTERIOLOGY 

carbolic  acid  in  solution.  A  3  or  4  per  cent,  solution  of 
commercial  carbolic  acid  in  water  requires  longer  for  disin- 
fection; but  it  is,  at  the  same  time,  open  to  fewer  objections 
than  are  solutions  of  the  inorganic  salts;  though  here,  too, 
we  find  a  somewhat  analogous  reaction  between  the  car- 
bolic acid  and  proteid  matters.  Under  ordinary  circum- 
stances its  action  is  compilete  in  from  twenty  minutes  to 
a,  .half-hour,,  .  :It  is  not  reliable  for  the  disinfection  of 
resistant  spores;  such,  for  instance,  as  those  of  bacillus 


All  tissues  containing  infectious  organisms  should  be 
burned,  and  all  cloths,  test-tubes,  flasks,  and  dishes  should 
be  bolied  in  2  per  cent,  soda  (ordinary  washing-soda)  solu- 
tion for  fifteen  to  twenty  minutes,  or  placed  in  the  steam 
sterilizer  for  half  an  hour. 

Intestinal  evacuations  may  best  be  disinfected  with 
boiling  water  or  with  milk  of  lime,  a  mixture  composed  of 
lime  in  solution  and  in  suspension  —  ordinary  fluid  "white 
wash."  This  should  be  thoroughly  mixed  with  the  evacua- 
tions until  the  mass  contains  a  considerable  excess  of  the 
lime,  and  should  remain  in  contact  with  them  for  one  or 
two  hours.  Excreta  may  also  be  easily  disinfected  by 
thoroughly  mixing  them  with  two  or  three  times  their 
volume  of  boiling  water,  after  which  they  are  kept  covered 
until  cool. 

Sputum  in  which  tubercle  bacilli  are  present,  as  well  as 
the  vessel  containing  it,  must  be  boiled  in  2  per  cent,  soda 
for  fifteen  minutes,  or  steamed  in  the  sterilizer  for  at  least 
a  half-hour. 

On  the  whole,  in  the  laboratory  we  should  rely  more  upon 
the  destructive  properties  of  heat  than  upon  those  of  chemical 
agents. 


CHEMICAL  STERILIZATION  AND  DISINFECTION     103 

From  what  has  been  said,  the  absurdity  of  sprinkling 
here  and  there  a  little  carbolic  acid,  or  of  placing  vessels 
of  carbolic  acid  about  apartments  in  which  infectious 
diseases  are  in  progress,  must  be  plain.  Treatment  of  water- 
closets  and  cesspools  by  allowing  now  and  then  a  few  cubic 
centimeters  of  some  so-called  disinfectant  to  trickle  through 
the  pipes  is  ridiculous.  A  disinfectant  must  be  applied  to 
the  bacteria,  and  must  be  in  contact  with  them  for  a  long  enough 
time  to  insure  the  destruction  of  their  life. 

In  the  light  of  the  latest  experiments  upon  disinfectants, 
the  place  formerly  occupied  by  many  agents  in  the  list  of 
substances  employed  for  the  purpose  will  most  likely  be 
changed  as  they  are  studied  more  closely.  The  agents, 
then,  which  will  prove  of  greatest  value  in  the  laboratory 
for  the  purpose  of  rendering  infectious  materials  harmless 
are:  heat,  either  by  burning,  by  steaming  for  from  half  an 
hour  to  an  hour,  or  by  boiling  in  a  2  per  cent,  sodium  car- 
bonate solution  for  fifteen  minutes;  3  to  4  per  cent,  solution 
of  commercial  carbolic  acid;  milk  of  lime,  and  a  solution  of 
chlorinated  lime  ("chloride  of  lime")  containing  not  less 
than  0.25  per  cent,  of  free  chlorine.  The  chloride  of  lime 
from  which  such  a  solution  is  to  be  made  should  be  fresh 
and  of  good  quality.  Good  chlorinated  lime,  as  purchased 
in  the  shops,  should  contain  not  less  than  25  to  30  per  cent, 
of  available  chlorine.  The  materials  to  be  disinfected  in 
either  of  the  lime  solutions  should  remain  in  them  for  about 
two  hours.  The  solutions  should  be  freshly  prepared  when 
needed,  as  they  rapidly  decompose  upon  standing. 


CHAPTER  IV. 

Principles  Involved  in  the  Methods  of  Isolation  of  Bacteria  in  Pure  Culture 
by  the  Plate  Method  of  Koch — Materials  Employed. 

As  was  stated  in  the  introductory  chapter,  the  isolation 
in  pure  cultures  of  the  different  species  that  may  be  present 
in  mixtures  of  bacteria  was  rendered  possible  only  through 
the  methods  suggested  by  Koch.  Since  the  adoption  of 
these  methods  they  have  undergone  many  modifications, 
but  the  fundamental  principle  remains  the  same.  The 
observation  that  lead  to  their  development  is  of  almost 
daily  occurrence.  When  bread,  cooked  potatoes  or  old  bits 
of  leather  are  left  in  moist,  damp  surroundings  they  invari- 
ably become  "moldy"  as  we  call  it;  that  is  to  say,  they 
become  more  or  less  covered  or  spotted  with  deposits  that 
are  known  to  be  composed  of  living  microorganisms.  . 

If  one  watches  the  evolution  of  this  condition  from  day 
to  day  it  will  be  seen  that  the  moldy  deposit  begins  as  a 
number  of  small  isolated  points  which,  as  they  get  larger, 
may  finally  coalesce  into  a  confluent  mass  that  eventually 
covers  the  surface.  If  one  examine  these  points,  however, 
before  they  begin  to  run  together,  it  is  found  that  they  are 
composed  of  microorganisms  of  several  different  kinds, 
some  being  molds,  some  yeasts,  and  some  bacteria.  The 
isolated  growths  of  these  various  species  present  different 
naked-eye  appearances,  so  that  even  at  a  glance  we  are 
justified  in  suspecting  that  they  are  of  a  'different  nature. 
They  develop  from  single  cells  that  have  fallen  upon  the 
(104) 


PRINCIPLES  IN  METHODS  OF  ISOLATION         105 

moist  objects  from  the  air,  and  as  the  cell  grows  and  mul- 
tiplies it  forms  these  circumscribed  patches  or  "colonies" 
as  they  are  called. 

*  The  question  that  then  presented  itself  was:  If  from  a 
mixture  of  organisms  floating  in  the  air  it  is  possible  in  this 
way  to  obtain  in  pure  cultures  the  component  individuals, 
what  means  can  be  employed  for  obtaining  the  same  results 
at  mil  from  mixture  of  different  species  of  bacteria  when 
found  together  under  other  conditions?  It  was  plain  that 
the  organisms  were  to  be  distinguished  primarily,  the  one 
from  the  other,  only  by  the  structure  and  general  appear- 
ance of  the  colonies  growing  from  them,  for  by  their  mor- 
phology alone  this  is  impossible.  What  means  might  be 
devised,  then,  fpr  separating  the  individual  members  of  a 
mixture  in  such  a  way  that  they  would  remain  in  a  fixed 
position,  and  be  so  widely  separated,  the  one  from  the  other, 
as  not  to  interfere  with  the  production  of  colonies  of  charac- 
teristic appearance,  which  would,  under  favorable  condi- 
tions, develop  from  each  individual  cell? 

If  one  take  in  the  hand  a  mixture  of  several  kinds  of 
flower  seeds  and  attempt  to  separate  the  mass  into  its  con- 
stituents by  picking  out  the  different  grains,  the  task  is 
tedious,  to  say  the  least  of  it;  but  if  the  handful  of  seeds 
be  thrown  upon  a  large  flat  surface,  as  upon  a  table,  the 
grains  become  widely  separated  and  the  matter  is  con- 
siderably simplified;  or,  if  sown  upon  proper  soil,  the  various 
grains  germinate  and  develop  into  plants  of  entirely  different 
characteristics,  by  which  they  can  readily  be  recognized 
as  distinct  species.  Similarly,  if  a  test-tube  of  decomposed 
bouillon  be  poured  upon  a  large,  flat  surface,  the  individual 
bacteria  in  the  mass  are  much  more  widely  separated,  the 
one  from  the  other,  than  they  were  when  the  bouillon  wfts 


106  BACTERIOLOGY 

in  the  tube;  but  they  are  in  a  fluid  medium,  and  there  is 
no  possibility  of  their  either  remaining  separated  or  of 
their  colonizing  under  these  conditions,  so  that  it  is  impos- 
sible by  this  means  to  pick  out  the  individuals  from  the 
mixture. 

FIG.  14 


Showing  certain  macroscopic  characteristics  of  colonies.    Natural  size. 

If,  however,  some  substance  can  be  found  which  possesses 
the  property  of  being  at  one  time  fluid  and  at  another  time 
solid,  and  which  can  be  added  to  this  bouillon  without  in 
any  way  interfering  with  the  life-functions  of  the  bacteria, 
then,  as  solidification  set  in,  the  organisms  would  be  fixed 


PRINCIPLES  IN  METHODS  OF  ISOLATION        107 

in  their  positions,  and  the  conditions  would  be  analogous 
to  those  seen  on  the  bits  of  potato,  bread  or  leather. 

Gelatin  possesses  this  property,  and  it  was,  therefore, 
used.  At  a  temperature  which  does  not  interfere  with  the 
life  of  the  bacteria  it  is  quite  fluid,  whereas  when  subjected 
to  a  lower  temperature  it  solidifies.  When  once  solid  it 
may  be  kept  at  a  temperature  favorable  to  the  growth  of 
the  bacteria  and  will  remain  in  its  solid  state. 

Gelatin  was  added  to  the  fluids  containing  mixtures  of 
bacteria,  and  the  whole  was  then  poured  upon  a  large,  flat 
surface,  allowed  to  solidify,  and  the  results  noted.  It  was 
found  that  the  conditions  seen  on  the  slice  of  moldy  potato 
could  be  reproduced;  that  the  indivduals  in  the  mixture 
of  bacteria  grew  well  in  the  gelatin,  and,  as  on  the  potato, 
grew  in  colonies  of  typical  macroscopic  peculiarities,  so 
that  they  could  easily  be  distinguished  the  one  from  the 
other  by  their  naked-eye  appearances.  (See  Fig.  14.)  It 
was  necessary,  however,  to  use  a  more  dilute  mixture  of 
bacteria  than  the  original  decomposed  bouillon.  The 
number  of  individuals  in  the  tube  was  so  enormous  that  on 
the  gelatin  plate  they  were  so  closely  packed  together  that 
it  was  impossible  to  pick  them  out,  not  only  because  of 
their  proximity  the  one  to  the  other,  but  also  because  this 
packing  together  materially  interfered  with  the  production 
of  those  characteristic  differences  visible  to  the  naked  eye. 
The  numbers  of  the  organisms  were  then  diminished  by  a 
process  of  dilution,  consisting  of  transferring  a  small  portion 
of  the  original  mixture  into  a  second  tube  of  sterilized  bouillon 
to  which  gelatin  had  been  added  and  liquefied;  from  this 
a  portion  was  added  to  a  third  gelatin-bouillon  tube,  and 
so  on.  These  were  then  poured  upon  large,  cold  surfaces 
and  allowed  to  solidify.  The  result  was  entirely  satisfactory. 


108  BACTERIOLOGY 

On  the  gelatin  plates  from  the  original  tube,  as  was  expected, 
the  colonies  were  too  numerous  to  be  of  use;  on  the  plates 
made  from  the  first  dilution  they  were  much  fewer  in  number, 
but  usually  they  were  still  too  numerous  and  too  closely 
packed  to  permit  of  characteristic  growth;  on  the  second 
dilution  they  were,  as  a  rule,  fewer  in  number  and  widely 
separated,  so  that  the  individuals  of  each  species  were  in 
no  way  prevented  by  the  proximity  of  their  neighbors  from 
growing  each  in  its  typical  way.  (See  Fig.  15.)  There 

FIG.  15 


Series  of  plates  showing  the  results  of  dilution  upon  the  number  of 
colonies:  A,  Plate  No.  1,  or  "original;"  B,  first  dilution,  or  Plate  No.  2; 
C,  second  dilution,  or  Plate  No.  3.  About  one-fourth  natural  size. 


was  then  no  difficulty  in  picking  out  the  colonies  resulting 
from  the  growth  of  the  different  individual  bacteria.  This, 
then,  is  the  principle  underlying  Koch's  method  for  the 
isolation  of  bacteria  by  means  of  solid  media. 

The  fundamental  constituent  of  the  media  employed  is 
the  bouillon,  which  contains  all  the  elements  necessary  for 
the  nutrition  of  most  bacteria,  the  gelatin  being  employed 
simply  for  the  purpose  of  rendering  the  bouillon  solid.  The 
medium  on  which  the  organisms  are  growing  is,  therefore, 
simply  solidified  bouillon,  or  beef -tea. 


PRINCIPLES  IN  METHODS  OF  ISOLATION        109 

In  practice  two  gelatinous  substances  are  employed — 
the  one  an  animal  or  bone  gelatin,  the  ordinary  table  gelatin 
of  good  quality;  the  other  a  vegetable  gum,  known  as 
agar-agar,  the  native  name  for  Ceylon  moss  or  Bengal 
isinglass,  which  is  obtained  from  a  group  of  marine  algse 
found  along  the  coast  of  Japan,  China,  and  many  parts 
of  the  East,  where  it  is  employed  as  an  article  of  diet  by  the 
natives. 

The  behavior  of  the  two  gelatinous  substances  under  the 
influence  of  heat  and  of  bacterial  growth  renders  them  of 
different  application  in  bacteriological  work.  The  animal 
gelatin  liquefies  at  a  much  lower  temperature,  and  also  re- 
quires a  lower  temperature  for  its  solidification,  than  does  the 
agar-agar.  Ordinary  gelatin,  in  the  proportion  commonly  used 
in  this  work,  liquefies  at  about  24°-26°  C.,  and  becomes  solid 
at  from  8°-10°  C.  It  may  be  employed  for  those  organisms 
which  do  not  require  a  higher  temperature  for  their  develop- 
ment than  22°-24°  C.  Agar-agar,  on  the  other  hand,  does 
not  liquefy  until  the  temperature  has  reached  about  98°-99° 
C.  It  remains  fluid  ordinarily  until  the  temperature  has 
fallen  to  38°-39°  C.,  when  it  rapidly  solidifies.  For  our 
purposes,  only  that  form  of  agar-agar  can  be  used  which 
remains  fluid  at  from  38°-40°  C.  Agar-agar  which  remains 
fluid  only  at  a  temperature  above  this  point  would  be  too 
hot,  when  in  a  fluid  state,  for  use;  many  of  the  organisms 
introduced  into  it  would  either  be  destroyed  or  checked  in 
their  development  by  so  high  a  temperature.  Agar-agar 
is  employed  in  those  cases  in  which  the  cultivation  must  be 
conducted  at  a  temperature  above  the  melting-point  of 
gelatin. 

In  addition  to  their  thermal  reactions,  these  two  gelati- 
nous substances  are  affected  very  differently  by  different 


110  BACTERIOLOGY 

species  of  bacteria.  As  was  said  above,  and  as  we  shall  soon 
see  for  ourselves,  certain  bacteria  elaborate  in  the  course  of 
their  growth  digestive  enzymes  or  ferments  that  in  their 
action  upon  proteid  matters  are  strikingly  like  pepsin  in 
some  and  trypsin  in  other  instances.  When  bacteria  en- 
dowed with  this  physiological  property  are  cultivated  upon 
bone  gelatin  their  growth  is  accompanied  by  the  progressive 
digestion  (liquefaction)  of  the  gelatin,  which  liquefied 
gelatin  cannot  again  be  brought  to  a  solid  condition.  We 
know  of  no  bacteria  capable  of  producing  a  similar  lique- 
faction of  agar-agar  or  vegetable  gum. 

As  a  rule,  the  colony-formations  seen  upon  gelatin  are 
much  more  characteristic  than  those  which  develop  on  agar- 
agar,  and  for  this  reason  gelatin  is  to  be  preferred  when 
circumstances  will  permit.  Both  gelatin  and  agar-agar  may 
be  used  for  the  isolation  of  species  from  mixtures. 


CHAPTER  V. 

Reactions — Methods  for  Adjustment — Titration — Hydrogen-ion  Concentra- 
tion— Preparation  of  Media — Bouillon,  Gelatin,  Agar-agar,  Potato, 
Blood  Serum,  Blood  Serum  from  Small  Animals,  Milk,  Litmus- whey 
Milk,  Durham's  Peptone  Solution,  Lactose  Litmus-agar,  Loffler's 
Blood-serum  Mixture,  the  Serum-water  Media  of  Hiss,  Guarniari's 
Gelatin-agar  Mixture. 


REACTION:— METHODS  OF  ADJUSTING. 

OF  fundamental  importance  to  the  successful  cultivation 
and  study  of  bacteria  upon  artificial  media  is  the  reaction 
of  the  media  used.  For  most  purposes  this  should  be  at 
or  about  neutral. 

Reaction  may  be  roughly  determined  by  the  use  of  litmus 
papers:  Acids  causing  the  blue  paper  to  turn  red  and  alka- 
lies turning  the  red  paper  blue. 

It  may  also  be  determined  by  titration,  the  indicator  used 
being  a  substance  that  announces  by  changes  in  color  slight 
deviations  to  the  acid  or  alkaline  side  of  neutral. 

It  may  also  be  determined  by  estimating  the  total  acidity 
as  indicated  by  the  hydrogen-ion  content  resulting  from 
the  dissociation  of  various  electrolytes  dissolved  in  the 
medium. 

For  a  long  time  the  simple  method  of  adjusting  the  reac- 
tion of  culture  media  by  the  use  of  litmus  papers  was  thought 
all  that  was  necessary.  Closer  study  of  the  matter,  however, 
revealed,  among  other  facts,  that  litmus  is  inconstant  in  its 
composition  and  that  it  also  undergoes  changes  in  color 
resulting  from  influences  other  than  those  of  acids  and 
alkalies.  In  consequence,  for  more  exact  work  other  methods 
have  been  developed. 

(ill) 


112  BACTERIOLOGY 

The  first  of  these  was  the  titration  of  a  known  volume 
of  the  culture  medium  with  alkaline  solutions  of  known 
strength  and  the  use  of  indicators  believed  at  the  time  to 
be  trustworthy  in  revealing  very  minute  deviations  from 
the  neutral  point.  From  the  amount  of  alkali  used  in  estab- 
lishing the  neutral  point  of  a  small  fraction  of  the  mass 
under  consideration  it  was  easily  possible  to  calculate  the 
amount  needed  for  the  whole. 

The  indicator  commonly  employed  in  such  titrations  is 
phenolphthalein,  a  compound  which  in  neutral  or  faintly 
acid  solutions  is  practically  colorless  but  which  shades  from 
a  delicate  pink  into  deep  magenta  as  alkalinity  gradually 
increases. 

The  adoption  of  still  more  refined  physical  methods  to 
the  study  of  solutions,  together  with  the  fact  laid  down 
by  Arrhenius  that  many  substances  when  dissolved  in 
water  no  longer  retain  their  molecular  structure  but  in 
part  or  whole  are  dissociated  into  atomic  modifications 
that  he  terms  ions,  some  of  which  carry  positive  and  others 
negative  electrical  charges,  have  led  to  a  complete  revision 
of  our  views  on  the  questions  of  "the  reactions  of  solutions" 
as  the  expression  had  hitherto  been  employed. 

As  a  result  of  all  this  two  general  plans  for  determining 
acidity  or  alkalinity  of  culture  media  are  now  in  use — the 
one  the  titration  method,  the  other  the  estimation  of  the 
aggregate  electro-positive  ions  present  in  the  solution,  i.  e., 
the  hydrogen-ion  concentration  of  the  solution.  As  there  is 
every  reason  for  regarding  the  latter  method  as  the  more 
exact,  it  is  likely  that  in  the  near  future  it  will  supersede  all 
others,  particularly  as  the  mode  of  its  application  is  becom- 
ing constantly  more  and  more  simplified  and  adapted  to  the 
routine  needs  of  the  bacteriologist. 


REACTION  AS  DETERMINED  BY  TITRATION      113 

REACTION  AS  DETERMINED  BY  TITRATION. 

In  the  development  of  this  method,  it  is  of  prime  impor- 
tance that  the  indicator  used  shall  announce  as  nearly  as 
possible  the  true  neutral  point,  i.  e.,  neutral  as  represented 
by  distilled  water.  Experience  with  three  well-known  indi- 
cators will  illustrate  the  importance  of  settling  this  point: 
If,  for  instance,  we  decide  to  establish  the  reaction  of  a 
volume  of  meat  infusion  agar-agar,  with  the  view  of  ulti- 
mately adjusting  that  reaction  to  neutral,  we  shall  first 
find  (as  with  practically  all  other  artificial  media)  that  the 
mass  is  acid.  If  we  now  undertake  to  determine  the  volume 
of  T  KOH  solution  that  is  necessary  to  neutralize  the  acid 
present,  we  shall  find  that  volume  to  vary  considerably 
according  to  the  indicator  employed.  If  phenolphthalein 
be  selected  as  the  indicator,  about  47  c.c.  of  the  alkaline 
solution  per  liter  of  culture  medium  will  be  required;  whereas, 
if  litmus  be  used  instead  of  phenolphthalein,  only  about 
28  c.c.  of  the  alkali  will  be  needed,  while  if  rosolic  acid  be 
substituted  for  litmus  the  figure  for  the  alkali  required  falls 
to  about  5  c.c.  Obviously,  if  our  information  ceased  here 
we  would  be  at  a  loss  to  know  just  which  of  the  three  titra- 
tions  represented  exact  neutralization  of  the  mass. 

Fortunately,  the  application  of  precise  physical  methods 
to  the  study  of  indicators  has  established  their  relative 
values  and  revealed  the  limits  of  their  usefulness  under 
various  circumstances.  As  a  result  of  such  studies  we  are 
informed  that  the  neutral  point  (the  reaction  of  pure  dis- 
tilled water)  as  announced  by  phenolphthalein  is  a  little 
high,  while  that  given  by  rosolic  acid  is  so  low  as  to  be 
altogether  misleading.  On  the  other  hand,  the  indications 
afforded  by  litmus  may  be  exact  or  nearly  so,  but  as  litmus 


114  BACTERIOLOGY 

is  not  a  stable  compound,  as  its  quality  and  sensitiveness 
is  subject  to  variations,  it  is  deemed  wisest  to  rely,  in  this 
method  upon  phenolphthalein,  its  high  neutral  point  being 
easily  adjusted  by  the  necessary  correction. 

The  method  of  titration  as  recommended  by  Fuller1  is 
essentially  as  follows:  5  c.c.  of  the  culture  medium  are  to 
be  mixed  with  45  c.c.  of  distilled  water  in  a  porcelain  evapo- 
rating dish  or  casserole  and  boiled  for  three  minutes,  after 
which  1  c.c.  of  a  phenolphthalein  solution  (0.5  per  cent,  in 
50  per  cent,  alcohol)  is  added  and  titration  with  •$-$•  KOH  is 
quickly  made.  The  neutral  point,  slightly  to  the  alkaline 
side,  is  announced  by  the  assumption  of  a  permanent,  pale 
pink  color,  the  effect  of  the  free  alkali  on  the  indicator. 
From  the  volume  of  ^  KOH  needed  to  neutralize  the  5  c.c. 
of  culture  medium,  one  can  readily  calculate  the  amount 
needed  for  the  whole  mass,  the  volume  of  which  must,  of 
course,  be  knowrn.  For  the  neutralization  of  the  entire  mass 
one  uses  not  •$$  KOH  but  y  KOH,  that  is,  an  alkaline  solu- 
tion twenty  times  as  strong  as  that  with  which  the  titration 
is  made  and  therefore  of  only  ^V  the  volume. 

To  illustrate :  If  to  neutralize  5  c.c.  of  a  nutrient  gelatin 
5.5  c.c.  of  ^r  KOH  are  required,  and  the  original  mass 
represented  by  the  5  c.c.  is  a  liter  in  volume,  manifestly 
the  whole  mass  would  require  two  hundred  times  5.5  c.c. 
or  1100  c.c.  of  the  alkaline  solution,  a  volume  much  too 
great  to  add  to  the  gelatin  because  of  the  extreme  dilution 
that  would  result,  we  therefore  substitute  for  the  YTT  KOH 
in  the  final  correction  the  normal  KOH  solution  ( f  KOH) ; 
which  being  twenty  times  as  strong  will  necessitate  the 
addition  of  only  -^V  of  the  volume,  that  is  ^y|p  =  55  c.c. 
of  f  KOH  for  the  liter. 

1  Jour.  Am.  Pub.  Health  Assn.,  Quarterly  Series,  1895,  p.  38L 


REACTION    BY  HYDROGEN-ION  CONCENTRATION     115 

On  the  average  the  neutral  point  as  established  by  this 
method  requires  for  a  liter  of  nutrient,  meat  infusion  agar- 
agar  the  addition  of  47  c.c.  T  KOH  and  for  a  liter  of  meat 
infusion  gelatin  56  c.c.  T  KOH.  Experience  shows  that 
media  neutral  to  phenolphthalein  are  somewhat  too  alkaline 
for  the  best  development  of  most  bacteria.  It  is  desirable 
therefore  to  make  certain  corrections.  In  Fuller's  experi- 
ence the  degree  of  deviation  from  phenolphthalein  neutral- 
ity that  insures  in  general  the  best  results  is  represented  by 
from  15  to  20  of  his  scale — that  is,  to  say  there  should 
remain  enough  uncombined  acid  in  a  liter  of  the  finished 
media  to  require  a  further  addition  of  from  15  to  20  c.c. 
T  KOH.  Thus  for  instance  if,  as  in  our  foregoing  calcula- 
tion 55  c.c.  T  KOH  were  needed  to  bring  the  mass  to 
phenolphthalein  neutrality,  we  would  actually  add  from  35 
to  40  c.c.,  i.  e.,  from  15  to  20  c.c.  less  than  was  indicated  by 
the  titration. 


REACTION  AS  DETERMINED  BY  HYDROGEN-ION 
CONCENTRATION. 

This  is  based  on  Arrhenius's  demonstration  that  when 
acids,  bases  and  salts  are  dissolved  in  water  their  molecules 
are  dissociated  into  electropositive  and  electronegative 
ions.  Not  all  the  molecules  of  such  electrolytes  are  always 
so  dissociated,  the  proportion  being  dependent  upon  a 
number  of  circumstances,  such  as  degree  of  dilution, 
temperature,  character  of  solvent,  etc.  By  appropriate 
electrical  methods  the  degree  of  such  dissociation  may  be 
accurately  discovered.  Analyses  made  in  this  manner  have 
afforded  results  of  fundamental  importance  to  an  understand- 
ing of  solutions,  of  acidity,  of  alkalinity  and  of  relative 
combining  values  of  dissolved  substances. 


'116  BACTERIOLOGY 

Of  prime  importance  to  the  bacteriologist  is  the  fact  that 
while  the  degree  of  dissociation  undergone  by  an  electro- 
lyte when  dissolved  in  pure  water  may  be  accurately  deter- 
mined and  is  constant  under  fixed  conditions,  if  such  electro- 
lyte be  dissolved  in  organic  fluids  the  estimation  of  the 
amount  of  dissociation  is  interfered  with  by  a  number  of 
organic  bodies  present.  Such  interfering  matters  are  known 
as  "buffers"  and  their  influence  must  always  be  taken  into 
account  in  this  method  of  estimating  reaction  of  a  fluid, 
i.  e.,  its  hydrogen-ion  concentration. 

The  meaning  of  the  term  "hydrogen-ion  concentration" 
can  best  be  understood  after  the  statement  of  several  funda- 
mental facts:  If  an  acid,  such  as  HC1,  for  instance,  be  dis- 
solved in  water,  some  of  it  retains  its  characteristic  mole- 
cular form  HC1,  but  a  much  larger  proportion  is  broken 
down,  "dissociated,"  into  its  component  elements  H  and  Cl, 
which  are  conceived  as  atoms  or  groups  of  atoms  denomi- 
nated electrically  charged  "ions;"  those  carry  ing  the  positive 
charge  being  the  hydrogen  (H)  ions,  those  carrying  the 
negative  charge  being  the  chlorine  (Cl)  ions,  or  "cations" 
and  "anions"  respectively.  According  to  this  theory  the 
acidity  of  a  fluid  (the  strength  of  the  acid  dissolved)  is  pro- 
portionate to  the  amount  of  dissociated  hydrogen  ions 
present,  and  not  to  the  amount  of  alkali  required  to  neutra- 
lize the  acid.  "Thus,  the  percentage  dissociation  or  the 
amount  of  H  ions  set  free  in  YT  HC1  solution  was  found 
(Talbot,  1908)  to  be  90  per  cent.,  while  that  of  A  acetic 
acid  is  1.4  per  cent.  Hydrochloric  acid  according  to  these 
findings  is  therefore  sixty-four  times  stronger  in  acidity 
that  acetic  acid,  although  10  c.c.  of  yV  of  either  acid  will 
require  a  similar  10  c.c.  portion  of  YTT  NaOH  to  neutralize 
it.  Accordingly  the  only  correct  method  of  measuring  the 


REACTION  BY  HYDROGEN-ION  CONCENTRATION     117 

acid  strength  of  a  solution  is  to  determine  the  amount  of 
free  H  ions  or  the  hydrogen-ion  concentration  (H.I.C.)  of 
that  solution,  and  not  to  determine  the  amount  of  y^  NaOH 
necessary  to  neutralize  that  solution."1 

From  the  foregoing  it  is  obviously  not  possible  to  deter- 
mine by  titration  with  an  alkaline  solution  if  the  acidity  of 
the  fluid  under  consideration  is  due  to  the  presence  of  a 
strong  or  a  weak  acid  or,  to  put  it  in  other  words,  to  the 
presence  of  acids  readily  dissociated  with  the  liberation  of 
large  amounts  of  H  ions  or  to  weak  acids  in  which  the  reverse 
is  the  case.  Total  acidity  can,  therefore,  only  be  deter- 
mined by  estimating  the  H  ion  concentration  of  the  fluid 
under  consideration. 

By  the  use  of  appropriate  electrical  devices  the  H.I.C.  of 
a  fluid  may  be  accurately  determined  and  its  variations,  as 
the  fluid  is  diluted,  may  also  be  exactly  detected,  even 
when  reduced  by  dilution  to  such  minute  traces  as  would 
be  expressed  by  unwieldy  decimals.  To  obviate  the  use  of 
such  unwieldy  decimals  the  symbol  pH  is  employed,  and  by 
suffixing  to  it  a  numeral  representing  the  successive  dilu- 
tions by  10  that  a  normal  solution  may  have  undergone,  we 
have  a  brief  and  handy  way  of  expressing  H.I.C.  Thus, 
for  instance,  pH5  would  symbolize  the  H.I.C.  of  a  0.00001 
N  (the  fifth  decimal  point)  solution,  while  pH7  (neutrality 
as  represented  by  distilled  water)  would  symbolize  the 
H.I.C.  of  an  acid  the  dilution  of  which  is  expressed  by 
0.0000001  N,  or  the  seventh  decimal  point.  We  see,  there- 
fore, that  in  the  use  of  the  short,  convenient  symbol  pH,  we 
have  a  means  of  noting  the  degree  of  dilution  of  an  acid 
which  corresponds  to  a  determined  H.I.C.  for  that  acid  in  that 

1  Medalia,  Jour.  Bacteriol.,   1920,  No.  5,  vol.  v,  p.  442. 


118  BACTERIOLOGY 

dilution;  pH,  therefore,  increases  as  H.I.C.  diminishes.  Thus: 

Tj-i  N  acid        TTO  N  acid         TTO  N  acid  TTI 

PH1  =  ~To^;  PH2  =  Too~;  PH3  =  ToTocr»  etc.,  or  pHl  = 


0.1  N  acid;  pH2  =  0.01  N  acid;  pH3  =  0.001  N  acid,  etc. 

Estimation  of  H.I.C.  —This  may  be  done  by  the  employ- 
ment of  either  electrolytic  or  colorimetric  methods.  The 
former  is  the  more  exact,  but  for  a  variety  of  reasons  is  less 
well  adapted  to  routine  bacteriological  work  than  is  the 
latter. 

By  the  former,  the  exact  electrical  method,  the  true 
acidity,  i.  e.,  the  H.I.C.  of  a  number  of  substances  in  known 
dilutions  has  been  accurately  determined.  These  "stand- 
ard solutions"  are  so  adjusted  that  in  known  dilutions 
they  can  be  made  to  represent  a  fairly  regular  range  of 
H.I.C.  from  extreme  concentration  to  almost  infinite  dilu- 
tion. When  thus  prepared  they  serve  as  objects  on  which 
to  establish  the  value  of  various  indicators  for  various 
H.I.C.  ranges,  for  no  single  indicator  will  function  through- 
out the  entire  series  of  dilutions;  that  which  gives  its  most 
trustworthy  indication  at  pH3,  for  instance,  would  be 
valueless  when  mixed  with  a  solution  the  H.I.C.  of  which  is 
symbolyzed  by  pH7. 

By  arranging,  therefore,  a  regular  succession  of  known 
dilutions  of  an  electrolyte  whose  pH  under  dilution  is  known, 
from  the  strongest  point  down  to  the  weakest,  i.  e.,  by 
having  each  step  in  the  range  only  &  the  strength  of  the 
preceding,  it  is  found  that  several  indicators  each  function- 
ing best  at  some  fixed  point  will  be  required  to  cover  the 
range  embraced  between  concentration  and  neutrality.  And 
also  that  as  we  pass  from  the  maximum  point  of  efficiency 
for  such  indicators  to  either  the  acid  side  of  the  scale,  that 
is  to  the  lessor  pH,  or  to  the  alkaline,  or  greater  pH,  the 
color  of  the  indicator  is  so  modified  as  to  serve  as  a  guide  for 
fairly  accurate  interpolations  between  the  periods. 


INDICATORS  AND  THEIR  EMPLOYMENT          119 

If,  for  instance,  we  so  dilute  any  N  acid  with  distilled 
water  that  each  successive  dilution  will  be  weakened  by  a 
tenfold  dilution  we  can  by  comparison  with  a  known  stand- 
ard solution  arrange  a  scale  extending,  say  from  pHl  to  pH8, 
and  by  trying  various  indicators  at  various  points  on  the 
range,  determine  at  which  point  each  indicator  functions 
best.  That,  in  fact,  is  what  has  been  done  and  it  is  in  that 
manner  that  the  colorimetric  estimation  of  H.I.C.  as  symbo- 
lyzed  by  pH  is  determined. 

INDICATORS  AND  THEIR  EMPLOYMENT. 

If  one  prepare  a  normal  solution  of  an  electrolyte  in  pure 
water  and  from  such  solution  prepare  successive  dilutions 
by  negative  multiples  of  ten,  it  is  possible,  as  said  above, 
by  appropriate  electrical  methods  to  detect  the  ions  result- 
ing from  dissociation  in  .dilutions  reaching  the  millionth  or 
the  billionth  part,  as  expressed  by  the  appropriate  decimals. 
If,  for  instance,  such  dilutions  be  prepared  for  a  NHC1 
solution,  we  shall  not  find  that  the  hydrogen-ion  content 
of  each  tenth  strength  of  solution  is  precisely  one-tenth  of 
that  of  the  next  stronger  dilution,  for  the  higher  the  dilution 
of  an  electrolyte,  the  greater  the  ionization,  i.  e.,  the  greater 
the  dissociation.  It  is  necessary,  therefore,  to  establish  for 
each  successive  tenth  dilution  the  H.I.C.,  as  symbolized  by 
pH  for  that  dilution,  and  it  is  found  that  in  the  interval 
between  such  dilutions  the  pH  gradually  increases  from  the 
low  to  the  high  dilutions  so  that  such  increase  may  also 
be  expressed  in  decimals — thus  a  substance  whose  pH  is 
between  5  and  6  may  be  expressed  as  pH5.5,  or  pH5.8  as 
the  case  may  be. 

When  a  solution  has  been  so  standardized  by  the  electrical 


120  BACTERIOLOGY 

method  that  the  gradations  of  its  pH  are  known,  it  is  the 
"standard"  solution  with  which  solutions  of  unknown  pH 
(or  H.I.C.)  are  to  be  compared  when  the  estimations  are 
made  by  the  colorimetric  method. 

Thus,  if  to  a  standard  solution  of  pH6  we  add  the  indi- 
cator which  exhibits  its  most  trustworthy  color  changes  at 
or  about  that  point,  changing  to  one  color  or  shade  as  we 
approach  the  alkaline  side,  i.  e.,  as  its  pH  ascends,  and  to 
another  color  or  shade  as  we  approach  the  acid  side,  i.  e., 
as  pH  descends,  it  is  observed  that  if  such  indicator  behave 
in  an  identical  manner  with  an  unknown  solution,  the  pH 
of  that  solution  is  probably  the  same  or  approximately  the 
same  as  that  of  the  standard  solution. 

fV 

By  a  creful  study  of  indicators  it  is  found,  as  said,  that 
for  various  pH  values  different  indicators  must  be  chosen, 
as  none  act  equally  well  throughout  all  dilutions. 

According  to  Clark  and  Lubs,  Medalia  and  others : 

Thymol  blue,  acid  range,  operates  best  between  pH1.2 
and  2.8. 

Brom  phenol  blue,  between  pH3  and  4.6. 

Methyl  red,  between  pH4.4  and  6. 

Brom  cresol  purple,  between  pH5.2  and  6.8. 

Brom  thymol  blue,  between  pH6  and  7.6. 

Phenol  red-,  between  pH6.8  and  84. 

Cresol  red,  between  pH7.2  and  8.8. 

Thymol  blue,  alkaline  range,  between  pH8  and  9.6. 

Method  of  Barnett  and  Chapman. — As  it  is  desirable  for 
routine  bacteriological  work  to  have  culture  media  at 
or  about  the  neutrality  of  distilled  water,  pH7,  it  is 
obvious  that  only  that  indicator  which  operates  best  at 
or  about  that  point  is  the  one  of  most  immediate  inter- 
est. Viewing  the  question  from  this  standpoint  Barnett 


INDICATORS  AND  THEIR  EMPLOYMENT         121 

and  Chapman  have  introduced  a  very  simple  method  that 
appears  to  meet  all  the  requirements.     The  following  are 
the  essentials  of  that  method:    For  the  preparation  of  cul- 
ture media,  which  are  to  be  neutral  or  nearly  so,  the  bacteri- 
ologist is  concerned  only  with  reactions  falling  between  pH7 
and  pH8,  and  as  phenolsulphonephthalein  (phenol  red)  indi- 
cates best  at  about  these  points  it  is  the  indicator  used. 
In  this  method  use  is  made  of  the  principle  of  superimposing 
the  two  extreme  colors  of  the  indicators  in  determining  the 
so-called  "half  transformation"  point.     Within  the  range 
of  its  transition  from  red  to  yellow  -we  may  regard  the 
observed  color  of  a  phenol  red  solution  as  composed  of  a 
definite  amount  of  red  plus  a  definite  amount  of  yellow,  and 
such  a  color  may  be  exactly  duplicated  by  superimposing 
the  extreme  red  and  the  extreme  yellow  of  the  indicator 
in  proper  concentrations.    Thus,  if  to  one  test-tube  we  add 
5  c.c.  of  dilute  acid  and  to  another  5  c.c.  of  dilute  alkali  and 
to  each  add  5  drops  of  phenol  red  solution,  a  bright  yellow 
will  be  produced  in  the  acid  tube  and  a  bright  red  in  the 
alkaline.     But  if  we  look  toward  the  light  through  both 
tubes,  a  color  will  be  observed  that  is  half-way  between 
yellow  and  red.     In  fact,  it  will  be  identical  with  the  color 
produced  by  10  drops  of  the  indicator  solution  in  5  c.c.  of  a 
standard  solution  having  a  pH  value  of  7.9.    This  is  the 
''half  transformation"  point,  and  is  a  definite  constant  for 
this  indicator.     But  if  instead  of  using  equal  amounts  of 
indicator  in  each  of  the  two  tubes  we  vary  the  partition 
of  the  10  drops  of  indicator  between  them,  then  by  super- 
imposing each  pair  and  viewing  them  by  transmitted  light, 
a  series  of  colors  will  be  observed  which  will  cover  the  range 
of  usefulness   of  this  particular  indicator.     Once   such   a 
series  is  standardized   ("calibrated")   against  solutions  of 


122  BACTERIOLOGY 

known  hydrogen-ion  concentration,  it  may  be  used  as  a 
standard  for  the  determination  of  unknown  reactions. 
Results  obtained  by  such  a  procedure,  the  phosphate  solu- 
tion of  Sorensen  being  employed  as  the  standard  of  com- 
parison, are  as  follows: 

RESULTS  WITH  PHENOL  RED. 

Acid  tubes.  Alkaline  tubes. 

Drops  of  phenol  Drops  of  phenol 

red  solution.  red  solution.  pH. 

9  1  =          6.9 

8  2  =         7.2 

7  3  7.5. 

6  4  =7.7 

5  5  =         7.9 

4  6  =         8.1 

Outline  of  procedure  -and  equipment  needed:     Apparatus 
and  chemicals: 

(a)  Clean  test-tubes  of  approximately  the  same  diameter. 

(b)  A  5  c.c.  and  a  1  c.c.  volume  pipette. 

(c)  A  medicine  dropper  drawn  out  to  a  fine  point. 

(d)  A  burette. 

(e)  Indicator  solution:     0.01  per  cent,  phenol  red  in 

distilled  water. 
;  (/)   /TT  KOH.       ' 
(g)  HC1  or  H2SO4. 

(h)  Test-tube  rack,  double  row  of  holes. 
Preparation  of  Standard  Color  Series. — Twelve  test-tubes 
are  placed  in  two  rows  of  six.  Into  each  tube  of  one  of 
the  rows  five  (5)  c.c.  of  dilute  alkali  are  placed  (the  ^ 
KOH  solution  may  be  used).  Into  each  tube  of  the  other 
row  five  (5)  c.c.  of  very  dilute  acid  are  placed  (1  drop  of 
concentrated  HC1  or  H2SO4  in  100  c.c.  distilled  water). 

Into  the  six  acid  tubes  9,  8,  7,  6,  5,  4  drops,  respectively, 
of  the  indicator  solution  are  placed.     Into  the  six  correspond- 


INDICATORS  AND  THEIR  EMPLOYMENT         123 

ing  alkali  tubes,  1,  2,  3,  4,  5,  6  drops  of  the  indicator  solution 
are  placed.  (If  the  dropper  be  held  vertically  all  drops  will 
be  of  practically  the  same  volume.) 

Each  pair  of  tubes  thus  contain  10  drops  of  the  indicator 
solution  between  them,  and  the  series  of  six  pairs,  when 
viewed  by  transmitted  light,  will  correspond  to  pH  values 
shown  in  the  foregoing  table. 

In  order  to  determine  the  hydrogen-ion  concentration  of 
an  unknown  solution  whose  reaction  lies  within  the  ranges 
pH6.9  and  pHS.l,  5  c.c.  of  it  are  placed  in  a  test-tube, 
10  drops  of  the  indicator  solution  are  added,  and  its  color 
is  compared  with  those  of  the  six  pairs  of  tubes;  its  H.I.C. 
being  the  same  as  that  pair  with  which  its  color  corresponds. 

When  this  unknown  solution  is  a  bacterial  culture  medium 
the  procedure  is  as  follows:  1  c.c.  of  the  medium  to  be 
titrated  is  added  to  4  c.c.  of  distilled  water  in  a  test-tube. 
Ten  drops  of  the  indicator  solution  are  then  added  and 
the  initial  reaction  is  determined  by  comparing  the  color  of 
the  mixture  with  each  of  the  six  pairs  of  standard  colors 
already  prepared.  From  this  initial  reaction,  usually  too 
acid,  the  desired  reaction  is  obtained  by  titration  with  the 
|^  NaOH  solution.  When  the  desired  reaction  for  1  c.c.  of 
the  medium  is  thus  obtained,  fifty  times  that  figure  in  y 
NaOH  (not  <nr  NaOH)  will  be  needed  for  a  liter  of  the 
medium. 

If  in  making  the  titrations  the  volume  of  fluid  in  the 
unknown  solution  is  greatly  increased  by  the  titrate,  then 
the  volumes  in  the  standard  solutions  must  be  correspond- 
ingly increased  by  the  addition  of  distilled  water.1 

1  For  details  see,  Clark  and  Lubs,  Jour.  Bacteriol.,  1917,  No.  2,  vol.  ii,  p. 
109.  Barnett  and  Chapman,  Jour.  Am.  Med.  Assn.,  1918,  No.  15,  vol.  Ixx,  p. 
1062.  Report  of  Committee  on  Descriptive  Chart,  Soc.  Am.  Bacteriologists, 
Jour.  Bacteriol.,  1919,  No.  2,  vol.  iv,  p.  119.  Medalia,  Jour.  Bacteriol., 
1920,  No.  5,  vol.  v,  p.  441. 


124  BACTERIOLOGY 

Bouillon. — As  has  been  stated,  the  fundamental  con- 
stituent of  culture  media  is  beef-tea  or  bouillon.  The 
directions  of  Koch  for  the  preparation  of  this  medium 
have  undergone  many  modifications  to  meet  special  cases; 
but  for  general  use  the  formula  now  employed  is  as  follows: 
500  grams  of  finely  chopped  lean  beef  free  from  fat  and 
tendons,  are  to  be  soaked  in  1  liter  of  water  for  twenty- 
four  hours,  during  which  time  the  mixture  is  to  remain  in 
an  ice-chest  or  to  be  otherwise  kept  at  a  low  temperature. 
At  the  end  of  twenty-four  hours  it  is  to  be  strained  through 
a  coarse  towel  and  pressed  until  a  liter  of  fluid  is  obtained. 
To  this  are  to  be  added  10  grams  (1  per  cent.)  of  dried 
peptone  and  5  grams  (0.5  per  cent.)  of  common  salt 
(NaCl).  It  is  then  to  be  rendered  neutral  or  very  slightly 
alkaline  by  one  of  the  foregoing  methods.  The  mixture  is 
then  placed  in  an  agateware  or  porcelain-lined  saucepan 
over  a  free  flame,  and  kept  at  the  boiling-point  until  all 
the  albumin  is  coagulated  and  the  fluid  portion  is  of  a 
clear,  pale  straw  color.  It  is  then  filtered  through  a  folded 
paper  filter  and  sterilized  by  steam. 

Not  infrequently  the  filtered  bouillon,  neutralized  and 
sterilized,  will  be  seen  to  contain  a  fine,  flocculent  precipi- 
tate. This  may  be  due  either  to  excess  of  alkalinity  or  to 
incomplete  precipitation  of  the  albumin.  The  former  may 
be  corrected  with  dilute  acetic  or  hydrochloric  acid,  and  the 
bouillon  again  boiled,  filtered,  and  sterilized;  or,  if  due 
to  the  latter  cause,  subsequent  boiling  and  filtration  usually 
result  in  ridding  the  bouillon  of  the  precipitate. 

Another  modification  now  generally  employed  is  in  the 
substitution  of  meat  extracts  for  chopped  meat  in  making 
the  bouillon.  Almost  any  of  the  meat  extracts  of  com- 
merce answer  the  purpose,  though  we  usually  employ 


PREPARATION  OF  GELATIN  125 

Liebig's.  It  is  used  in  the  strength  of  from  two  to  four 
grams  to  the  liter  of  water.  Peptone  and  sodium  chloride 
are  added  as  in  the  bouillon  made  from  meat-infusion. 
The  advantages  of  meat  extract  are:  it  takes  less  time; 
affords  a  solution  of  more  uniform  composition  if  used  in 
fixed  proportions';  and  in  general  use  gives  results  that  are 
equally  as  satisfactory  as  those  obtained  from  the  employ- 
ment of  infusion  of  meat.  The  disadvantage  is  the  possible 
presence  of  antiseptics  or  preservatives. 

Nutrient  Gelatin. — For  the  preparation  of  gelatin  the 
bouillon  is  first  made  in  the  way  given,  except  that  its 
reaction  is  corrected  after  the  gelatin  has  been  completely 
dissolved,  which  occurs  very  rapidly  in  hot  bouillon.  The 
reaction  of  the  gelatin  of  commerce  is  frequently  more  or 
less  acid,  so  that  a  much  larger  amount  of  alkali  is  needed 
for  its  neutralization  than  for  other  media.  It  is  possible, 
however,  to  obtain  from  the  makers  an  excellent  grade 
of  gelatin  from  which  practically  all  free  acid  has  been 
carefully  washed.  The  gelatin  is  added  in  the  proportion 
of  10  to  12  per  cent.  Its  complete  solution  may  be  accom- 
plished either  over  a  water-bath,  in  the  steam  sterilizer,  or 
over  a  free  flame.  If  the  latter  method  be  practised,  care 
must  be  taken  that  the  mixture  is  constantly  stirred  to 
prevent  burning  at  the  bottom. 

It  is  now  almost  the  universal  practice  to  use  enamelled 
iron  saucepans,  instead  of  glass  vessels  for  the  purpose  of 
making  both  gelatin  and  agar-agar;  by  this  means  the 
free  flame  may  be  employed  without  danger  of  breaking  the 
vessel,  and,  with  a  little  care,  without  burning  the  media. 
Under  any  conditions  it  is  better  to  protect  the  bottom  of 
the  vessel  from  the  direct  action  of  the  flame  by  the  inter- 
position of  several  layers  of  wire  gauze,  a  thin  sheet  of  asbes- 
tos-board, or  an  ordinary  cast-iron  stove-plate. 


126 


BACTERIOLOGY 


When  the  gelatin  is  completely  melted  it  may  be  filtered 
through  a  folded  paper  filter  supported  on  an  ordinary 
funnel;  if  solution  is  complete,  this  should  be  very  quickly 
accomplished. 

To  Fold  a  Filter. — For  the  filtration  of  such  substances 
as  gelatin  and  agar-agar  it  is  of  mportance  to  have  a 
properly  folded  filter.  •  Inability  to  fold  a  filter  properly  is 
so  common  with  beginners  that  a  detailed  description  of 
the  steps  may  not  be  out  of  place.  To  fold  a  filter  cor- 
rectly, proceed  as  follows:  A  circular  piece  of  filter  paper 
is  folded  exactly  through  its  center,  forming  the  fold  1,  1' 


(Fig.  16);  the  end  1  is  then  folded  over  to  1',  forming  the 
fold  5;  1  and  1'  are  each  then  brought  to  5,  thus  forming 
the  folds  3  and  7;  1  is  then  carried  to  the  point  7,  and  the 
fold  4  is  formed,  and  by  carrying  V  to  3  the  fold  6  is  pro- 
duced; and  by  bringing  1  to  3  and  1'  to  7  the  folds  2  and  8 
result. 

Thus  far  the  ridges  of  all  folds  are  on  the  side  of  the  paper 
next  to  the  table  on  which  we  are  folding.  The  paper  is 
now  taken  up  and  each  space  between  the  seams  just  pro- 
duced is  to  be  subdivided  by  a  crease  or  fold  through  its 
center,  as  indicated  by  the  dotted  lines  in  Fig.  16,  but  with 


FOLDING  A  FILTER  127 

the  creases  on  the  side  opposite  to  that  occupied  by  creases 
1,  2,  3,  4,  etc.,  first  made.  As  each  of  these  folds  is  made 
the  paper  is  gradually  brought  into  a  wedge-shaped  bundle 
(Fig.  17,  a),  which  when  opened  assumes  the  form  of  a 
properly  folded  filter  (seen  in  6,  Fig.  17).  Before  placing 
it  upon  the  funnel  it  is  well  to  go  over  each  crease  and  see 
that  it  is  as  closely  folded  as  possible,  care  being  taken  not 
to  tear  it.  The  advantage  of  the  folded  filter  is  that  by  its 
use  a  much  greater  filtering,  surface  is  obtained,  as  it  is  in 
contact  with  the  funnel  only  at  the  points  formed  by  the 
ridges,  leaving  the  greater  part  of  the  flat  surface  free  for 

filtration. 

FIG.  17 


The  employment  of  the  hot-water  funnel,  so  often  recom- 
mended, has  been  dispensed  with  in  this  work  to  a  very 
large  extent,  for  the  reason  that  if  solution  of  the  gelatin 
is  complete,  filtration  is  so  rapid  as  not  to  necessitate  the 
use  of  an  apparatus  for  maintaining  a  high  temperature. 
The  temperature  at  which  the  hot-water  funnel  retains  the 
gelatin  is  so  high  that  evaporation  and  concentration  rapidly 
occur,  and  in  consequence  filtration  is,  as  a  rule,  retarded. 
The  filtration  is  frequently  done  in  the  steam  sterilizer; 
but  this,  too,  is  unnecessary  if  the  gelatin  is  quite  dissolved. 
At  the  ordinary  temperature  of  the  room,  and  by  the  means 
commonly  employed  for  the  filtration  of  other  substances, 


128  BACTERIOLOGY 

both  gelatin  and  agar-agar  may  be  rapidly  filtered  if  they 
are  completely  dissolved. 

It  not  infrequently  occurs  that,  even  under  the  most 
'careful  treatment,  the  filtered  gelatin  is  not  quite  trans- 
parent, and  clarification  becomes  necessary.  For  this 
purpose  the  mass  must  be  redissolved,  and  when  at  a  tem- 
perature between  60°  and  70°  C.  an  egg,  which  has  been 
beaten  up  with  about  50  c.c.  of  water,  is  added.  The  whole 
is  then  thoroughly  mixed  together  and  again  brought  to  the 
boiling-point,  and  kept  there  until  coagulation  of  the 
albumin  occurs.  The  egg  albumin  coagulates  as  large  floccu- 
lent  masses,  and  it  is  better  not  to  break  them  up,  as  when 
broken  up  into  fine  flakes  they  clog  the  filter  and  materially 
retard  filtration. 

The  practice  sometimes  recommended  of  removing  these 
albuminous  coagula  by  first  filtering  the  gelatin  through  a 
cloth,  and  then  through  paper,  is  not  only  superfluous,  but 
in  most  instances  renders  the  process  of  filtration  much  more 
difficult,  because  of  the  disintegration  of  the  masses  into 
finer  particles,  which  have  the  effect  just  mentioned,  viz., 
of  clogging  the  filter. 

Under  no  circumstances  should  a  filter  be  used  without 
first  having  been  moistened  with  water.  If  this  is  not  done 
the  pores  of  the  paper,  which  are  relatively  large  when  in  a 
dry  state,  when  moistened  by  the  gelatin  not  only  diminish 
in  size,  but  in  contracting  are  often  entirely  occluded  by  the 
finer  albuminous  flakes  which  become  fixed  within  them, 
and  filtration  practically  ceases.  The  preliminary  moisten- 
ing with  water  causes  diminution  of  the  size  of  the  pores  to 
such  an  extent  that  the  finer  particles  of  the  precipitate  rest 
on  the  surface  of  the  paper,  instead  of  becoming  fixed  in  its 
meshes. 


PREPARATION  OF  NUTRIENT  AGAR-AGAR        129 

During  boiling  it  is  well  to  filter,  from  time  to  time,  a 
''  few  cubic  centimeters  of  the  gelatin  into  a  test-tube  and  boil 
it  over  a  free  flame  for  a  minute  or  so;  in  this  way  one  can 
detect  if  all  the  albumin  has  been  coagulated — i.  e.,  if  the 
solution  is  ready  for  filtration. 

Gelatin  should  not,  as  a  rule,  be  boiled  more  than  ten  or 
fifteen  minutes  at  one  time,  or  be  left  in  the  steam  sterilizer 
for  more  than  thirty  minutes ;  otherwise  its  property  of 
solidifying  may  be  impaired. 

As  soon  as  the  preparation  of  the  gelatin  is  complete, 
whether  it  is  retained  in  the  flask  into  which  it  has  been 
filtered  or  decanted  into  sterilized  test-tubes,  it  should  be 
sterilized,  the  mouth  of  the  flask  or  the  test-tubes  containing 
it  having  been  previously  closed  with  cotton  plugs.  It  may 
be  sterilized  by  either  the  intermittent  method  with  stream- 
ing steam  or  by  a  single  application  of  steam  under  pressure 
in  the  autoclave.  If  the  latter  method  be  selected,  the 
pressure  should  not  exceed  one  atmosphere  and  the  time 
of  exposure  be  not  over  fifteen  minutes. 

Nutrient  Agar-agar. — The  preparation  of  nutrient  agar- 
agar  by  the  beginner  is  far  too  frequently  a  tedious  and 
time-consuming  operation.  This  is  due  mainly  to  lack  of 
patience  and  to  deviation  from  the  rules  laid  down  for 
the  preparation  of  this  medium.  If  the  directions  given 
below  for  the  preparation  of  nutrient  agar-agar  be  strictly 
observed,  no  difficulty  whatever  should  be  encountered. 
Many  methods  are  recommended  for  its  preparation,  almost 
every  worker  having  some  slight  modification  of  his  own. 

The  methods  that  have  given  us  the  best  results,  and  from 
which  we  have  no  good  grounds  for  departing,  are  as  follows : 

Prepare  the  bouillon  in  the  usual  way.  Agar-agar  reacts 
neutral  or  Very  slightly  alkaline,  so  that  the  bouillon  may 
9 


130  BACTERIOLOGY 

be  neutralized  before  the  agar-agar  is  added.  Then  add 
finely  chopped  or  powdered  agar-agar  in  the  proportion  of 
1  to  1.5  per  cent.  Place  the  mixture  in  a  porcelain-lined 
iron  vessel,  and  on  the  side  of  the  vessel  make  a  mark  at 
the  height  at  which  the  level  of  the  fluid  stands;  if  a  liter 
of  medium  is  being  made,  add  about  250  to  300  c.c.  more  of 
water  and  allow  the  mass  to  boil  slowly,  occasionally  stirring, 
over  a  free  flame,  from  one  and  a  half  to  two  hours;  or  until 
the  excess  of  water — i.  e.,  the  250  or  300  c.c.  that  were 
added — has  evaporated.  Care  must  be  taken  that  the 
mixture  does  not  boil  over  the  sides  of  the  vessel.  From  time 
to  time  observe  if  the  fluid  has  fallen  below  its  original 
level;  if  it  has,  add  hot  water  until  its  volume  of  1  liter  is 
restored.  At  the  end  of  the  time  given  remove  the  flame 
and  place  the  vessel  containing  the  mixture  in  a  large  dish 
of  cold  water;  stir  the  agar-agar  continuously  until  it  has 
cooled  to  about  68°-70°  C.,  and  then  add  the  white  of  one 
egg  which  has  been  beaten  up  on  about  50  c.c.  of  water; 
or  the  ordinary  dried  albumin  of  commerce  may  be  dissolved 
in  cold  water  in  the  proportion  of  about  10  per  cent,  and  used; 
the  results  are  equally  as  good  as  when  eggs  are  employed. 
Mix  this  carefully  throughout  the  agar-agar  and  allow  the 
mass  to  boil  slowly  for  about  another  half-hour,  observing 
all  the  while  the  level  of  the  fluid,  which  should  not  fall 
below  the  liter  mark.  It  is  necessary  to  reduce  the  tempera- 
ture of  the  mass  to  the  point  given,  68°-70°  C.,  otherwise 
the  coagulation  of  the  albumin  will  occur  suddenly  in  lumps 
and  masses  as  soon  as  it  is 'added,  and  its  clarifying  action 
will  not  be  uniform.  The  process  of  clarification  with  the 
egg  is  purely  mechanical;  the  finer  particles,  which  would 
otherwise  pass  through  the  pores  of  the  filter,  being  taken 
up  by  the  albumin  as  it  coagulates  and  retained  in  the 
coagula. 


FILTERING  AGAR-AGAR  131 

At  the  end  of  a  half-hour  the  boiling  mass  may  be  easily 
and  quickly  filtered  through  a  heavy,  folded  paper  filter 
at  the  room  temperature;  as  a  rule  the  filtrate  is  as  clear  and 
transparent  as  agar-agar  usually  appears. 

It  may  be  well  to  emphasize  the  fact  that  for  the  filtration 
of  agar-agar  no  special  device  for  maintaining  the  tem- 
perature of  the  mass,  is  necessary.  Agar-agar  prepared 
after  the  methods  just  given  should  pass  through  a  properly 
folded  paper  filter  at  the  rate  of  a  liter  in  from  twelve  to 
fifteen  minutes. 

Another  plan  that  insures  complete  solution  of  the  agar- 
agar  without  causing  the  precipitates  often  seen  when  all 
the  ingredients  are  added  at  once  and  boiled  for  a  long  time 
is  to  weigh  out  the  necessary  amount  of  agar-agar,  10  or  15 
grams,  and  place  this  in  1300  or  1400  c.c.  of  water  and  boil 
down  over  a  free  flame  to  1000  c.c.  The  peptone,  salt,  and 
beef  extract  are  then  added  and  the  boiling  continued  until 
they  are  dissolved.  The  clarification  with  egg-albumen  may 
then  be  done,  and  usually  the  mass  filters  quite  clear  and 
does  not  show  the  presence  of  precipitates  upon  cooling. 
If  the  mixture  is  positively  alkaline,  it  is  not  only  cloudy, 
but  it  filters  with  difficulty;  if  it  is  acid,  it  is  usually  quite 
clear,  and  filters  more  quickly,  but,  as  Schultz  has  pointed 
out,  it  loses  at  the  same  time  some  of  its  gelatinizing 
properties. 

Another  method  by  which  agar-agar  can  be  easily  and 
quickly  melted  is  by  steam  under  pressure.  If  the  flask 
containing  the  mixture  of  bouillon  and  agar-agar  be  kept 
in  the  digester  or  autoclave  for  ten  minutes  with  the  steam 
under  a  pressure  of  about  one  atmosphere,  as  shown  by  the 
gauge,  the  agar-agar  will  be  found  at  the  end  of  this  time 
completely  melted,  and  filtration  may  then  be  accomplished 
with  but  little  difficulty. 


132 


BACTERIOLOGY 


FIG.  18 


If  glycerin  is  to  be  added  to  the  agar-agar,  it  is  done  after 
filtration  and  before  sterilization.  The  nutritive  properties 
of  the  media  for  certain  organisms,  particularly  the  tubercle 
bacillus,  are  increased  by  the  addition  of  glycerin  in  the 
proportion  of  5  to  7  per  cent. 

If  after  filtration  a  fine  flocculent  precipitate  is  seen,  look 
to  the  reaction  of  the  medium.  If  it  is  quite  alkaline,  boilj 
neutralize,  and  filter  again.  If  the  reac- 
tion is  neutral  or  only  very  slightly  acid, 
dissolve  and  again  clarify  with  egg-albu- 
men by  the  method  given. 

The  most  important  feature  of  all  the 
media,  aside  from  the   correct  proportion 
of   the   ingredients,   is  their  reaction.    It 
must  be  neutral  or  very  slightly  alkaline 
to  litmus.    (See  remarks  on  Neutralization 
of  Media.)    Only  a  few  organisms  develop 
well  on  media  of  an  acid  reaction. 
*    Preparation   of  Potatoes. — With  an  ordi- 
nary cork  borer  punch  out  from  sound 
potatoes    cylindrical    bits    that    will   slip 
easily  into  the  test-tubes  to  be  used.     Cut 
away  all  particles  of  the  skin.    Then  cut 
on  each  cylinder  a  slanting  surface  extend- 
Potato  in  test-tube,     ing  from  about  the  middle  diagonally  to 
the  end.    Leave  the  cylinders  in  running 
water   over    night   to  prevent  them   from   becoming  dis- 
colored when  they  are  sterilized. 

One  potato  cylinder  thus  prepared  is  then  to  be  placed 
in  each  of  the  already  cleaned,  plugged  and  sterilized  test- 
tubes,  after  which  they  are  sterilized  by  either  the  inter- 
mittent method  with  streaming  steam  or  by  steam  under 


POTATOES  AND  BLOOD  SERUM  133 

pressure  in  the  autoclave.  In  the  latter  event  one  atmos- 
phere of  pressure  should  be  continued  for  twenty  minutes. 
(See  Fig.  18.) 

For  some  purposes  potatoes  may  be  advantageously 
peeled,  sliced  into  disks  of  about  1  cm.  in  thickness,  and 
placed  in  small  glass  dishes  provided  with  covers,  similar  to 
the  ordinary  crystallizing  dishes.  The  dish  and  its  con- 
tents are  then  sterilized  by  steam  in  the  usual  way.  By 
this  plan  a  relatively  large  area  for  cultivation  is  obtained. 

Potatoes  may  also  be  boiled,  or  steamed,  and  mashed, 
and  the  mass  placed  in  covered  dishes,  test-tubes,  or  flasks, 
and  sterilized.  By  this  method  one  obtains  in  the  mass  a 
mean  of  the  composition  of  the  several  potatoes,  or  bits  of 
potatoes,  used  in  making  it,  an  advantage  where  uniformity 
is  desired. 

Care  must  be  given  to  the  sterilization  of  potatoes,  because 
they  always  have  adhering  to  them  the  organisms  commonly 
found  in  the  ground,  the  spores  of  which  are  among  the 
most  resistant  known. 

Blood  Serum. — For  ordinary  routine  work  blood  serum 
may  be  obtained  from  either  the  slaughter  houses  or  the 
antitoxin  manufacturers.  When  from  the  former  the 
blood  that  streams  from  the  severed  vessels  of  the  throat 
of  the  slaughtered  animal  is  collected  under  as  clean  con- 
ditions as  possible  in  large,  clean  glass  museum  jars.  These 
are  then,  with  the  covers  placed  upon  them,  set  aside  in  an 
ice-chest  until  coagulation  is  complete.  The  serum  may  then 
be  decanted  or  pipetted  off  into  flasks  and  thus  transported 
to  the  laboratory  to  be  sterilized  by  the  method  given 
below. 

In  many  localities  it  is  possible  to  purchase  at  a  small 
cost  normal  horse  serum  in  bulk  from  firms  engaged  in  the 


134  BACTERIOLOGY 

manufacture  of  antitoxins  and  other  biological  products. 
This  serum,  obtained  under  aseptic  precautions,  has  ob- 
viously an  advantage,  and  has  in  our  hands  proven  entirely 
satisfactory  for  routine  work. 

In  either  case  the  serum  is  to  be  decanted  into  clean, 
sterile  test-tubes  provided  with  cotton  plugs,  after  which 
it  must  be  immediately  sterilized.  For  this  purpose  the 
method  suggested  by  Councilman  and  Mallory  is  now 

FIG.  19 


Chamber  for  sterilizing  and  solidifying  blood  serum.    (Koch.) 

generally  used.  It  is  as  follows:  Place  the  test-tubes  con- 
taining the  serum  in  a  slanting  position  in  a  dry  air  sterilizer 
and  heat  them  to  from  80°-90°  C.  for  a  time  necessary 
to  solidify  the  serum.  After  this  they  are  kept  for  twenty 
minutes  on  three  successive  days  in  the  steam  sterilizer  at 
100°  C.  They  should  be  kept  at  room  temperature  between 
the  exposures  to  the  steam.  After  this  treatment  the  serum 
should  be  sterile. 


BLOOD  SERUM  135 

Serum  thus  prepared  may  be  kept  from  drying  by  burning 
off  in  the  gas  flame  the  excess  of  cotton  protruding  from  the 
ends  of  the  tubes  and  then  forcing  down  upon  the  cotton 
plugs  clean,  new,  corks  that  have  been  sterilized  by  steam 
under  pressure.  (Ghriskey.) 

To  secure  satisfactory  results  by  this  method  several 
precautions  should  be  noted,  viz.:  The  solidification  of  the 
serum  in  the  dry  air  sterilizers  must  be  complete,  else  its 
surface  will  be  rough  and  broken  by  bubbles;  the  same 
results  if  the  temperature  in  the  dry  air  sterilizer  is  brought 
up  too  rapidly. 

Serum  prepared  in  this  way  is  neither  clear  nor  colorless. 
This  is  ordinarily  not  a  disadvantage.  The  popularity  of 
the  method  is  due  to  its  simplicity,  the  rapidity  with  which 
a  satisfactory  serum  may  be  prepared  and  especially  to 
the  fact  that  the  rigid  precautions  against  contamination 
observed  in  the  older  methods,  where  sterilization  at  low 
temperature  was  practised,  are  not  essential  to  success, 
since  even  though  such  contaminations  occur  they  are 
eliminated  by  the  high  temperatures  used  in  this  procedure. 

Blood  Serum  from  Small  Animals. — For  special  purposes 
it  is  often  desirable  to  secure  blood  serum  under  strictly 
aseptic  precautions  from  particular  species  of  animals, 
many  of  them  being  small.  To  this  end  there  have  been 
devised  a  number  of  handy  methods.  That  which  in  our 
hands  has  proven  the  simplest  and  generally  most  useful 
is  the  Rivas  modification1  of  Latapie's  method.  It  is  as 
follows: 

The  Rivas  apparatus  is  constructed  from  two  test-tubes 
about  15  x  180  mm.  in  size.  The  mouth  of  one  test-tube  is 
drawn  out  into  a  long  narrow  neck  1  cm.  in  diameter  and 

1  University  of  Pennsylvania,  Medical  Bulletin,  1904,  vol.  xvii,  p.  295. 


136 


BACTERIOLOGY 


about  5  cm.  in  length.    Three  or  four  points  on  the  side  of 
the  tube  are  softened  in  the  flame  of  a  blowpipe,  and  the 


FIG.  20 


Rivas  apparatus  for  collecting  blood  serum:  A,  long  narrow  neck  on 
first  tube;  B,  constriction  on  tubes  near  mouth;  C,  invaginations  on  first 
tube;  D,  small  cannula  drawn  out  on  extremity  of  first  tube;  E,  blood- 
clot,  and  F,  blood  serum  collected  in  bottom  of  second  tube. 


softened  glass  driven  inward  by  means  of  a  piece  of  pointed  > 
wood.    This  gives  supports  on  the  interior  of  the  tube  to 


BLOOD  SERUM  137 

hold  the  coagulated  blood  in  place.  Between  the  long 
narrow  neck  and  the  body  of  the  tube  a  constriction  is 
formed  by  drawing  out  the  tube  while  heated.  The  second 
tube  also  has  a  similar  constriction  about  20  cm.  from  its 
mouth. 

The  two  tubes  are  now  fitted  together  by  inserting  the 
one  with  the  long  narrow  neck  into  the  second  tube ;  a  small 
amount  of  cotton  being  first  carefully  folded  around  the 
neck  of  the  first  tube,  so  as  to  prevent  the  entrance  of  dust. 
The  two  tubes  are  then  fastened  together  by  means  of  a 
wire  twisted  around  the  constriction  at  the  neck  of  each 
tube,  and  the  apparatus  is  then  wrapped  in  cotton  and 
sterilized  in  a  hot-air  sterilizer. 

Before  using  the  apparatus  the  extremity  of  the  first  tube 
is  heated  in  the  gas-flame,  and  by  touching  this  point  with 
a  piece  of  pointed  glass  rod  it  is  gently  drawn  out  into  a 
fine  cannula.  When  the  animal  has  been  prepared  for  the 
operation  and  a  vessel  exposed,  the  point  of  the  cannula  is 
snipped  off  with  a  sterile  scissors,  when  the  point  of  the 
cannula  is  inserted  into  the  vessel.  The  pressure  of  blood 
is  sufficient  to  fill  the  first  tube.  The  point  of  the  cannula 
is  now  removed  from  the  vessel  and  sealed  in  a  gas-flame. 
The  apparatus  is  laid  aside  in  an  almost  horizontal  position 
until  the  blood  has  become  completely  coagulated.  It  is 
then  inverted  and  set  aside  for  the  serum  to  separate  and 
trickle  down  through  the  narrow  neck  of  the  first  tube  and 
collect  in  the  second  tube.  When  this  has  occurred,  the  wire 
holding  the  two  tubes  together  is  unwound,  and  the  first 
tube  is  removed  and  the  second  plugged  with  a  well-fitting 
sterile  cotton  plug,  when  the  serum  may  be  preserved  in 
the  tube  for  several  days  without  danger  of  contamination. 


138  BACTERIOLOGY 

Preservation  of  Blood  Serum. — It  is  sometimes  desirable 
to  preserve  blood  serum  in  a  fluid  state.  This  can  be  done 
by  the  fractional  method  of  sterilization  at  low  tempera- 
tures, already  described,  or  with  much  less  effort,  and  with- 
out the  use  of  heat,  by  a  method  that  we  have  found  very 
satisfactory.  In  the  course  of  Kirschner's  investigations 
chloroform  was  shown  to  possess  decided  disinfectant 
properties;  as  it  is  quite  volatile,  it  is  easily  got  rid  of  when 
its  disinfectant  or  antiseptic  properties  are  no  longer  required. 
If,  therefore,  the  serum  to  be  preserved  be  placed  in  a  closely 
stoppered  flask  and  enough  chloroform  added  to  form  a 
thin  layer,  about  2  mm.,  on  the  bottom,  the  serum  may 
be  kept  indefinitely  without  contamination,  so  long  as  the 
chloroform  is  not  permitted  to  evaporate.  This  latter  pro- 
vision is  one  on  which  success  depends.  If  the  vessel  con- 
taining the  mixture  of  chloroform  and  serum  be  not  tightly 
corked,  the  chloroform  vapor  escapes  pretty  rapidly  and 
exerts  no  preservative  action.  In  fact,  bacteria  will  grow 
uninterruptedly  in  a  cotton-stoppered  test-tube  containing 
bouillon  to  which  chloroform  has  been  added.  When  re- 
quired for  use,  the  serum  is  decanted  into  test-tubes,  which 
are  then  placed  in  a  water-bath  at  about  50°  C.  until  all 
the  chloroform  has  been  driven  off;  this  can  be  determined 
by  the  absence  of  its  characteristic  odor.  The  serum  may 
then  be  solidified,  sterilized  by  heat,  and  employed  for 
culture  purposes.  We  have  found  serum  so  preserved  to 
answer  all  requirements  as  a  culture  medium. 

Milk. — Fresh  milk  should  be  allowed  to  stand  overnight  in 
an  ice-chest,  the  cream  then  removed,  and  the  remainder  of 
the  milk  pipetted  into  test-tubes,  about  8  c.c.  to  each  tube, 
and  sterilized  by  the  intermittent  process,  at  the  tem- 
perature of  steam,  for  three  successive  days. 


MILK  139 

The  separation  of  the  cream  may  be  accelerated  and 
rendered  more  complete  if  the  cylinder  containing  the  milk 
be  placed  in  the  steam  sterilizer  for  fifteen  minutes  before  it 
is  placed  in  the  ice-chest. 

The  cream  is  best  separated  from  the  milk  by  the  use  of 
a  cylindrical  vessel  with  a  stopcock  at  the  bottom,  by 
means  of  which  the  milk,  devoid  of  cream,  may  be  drawn 
off.  A  Chevalier  creamometer  with  a  stopcock  at  the  bottom 
serves  the  purpose  very  well.  It  should  be  covered  while 
standing.1 

Milk  may  be  used  as  a  culture  medium  without  any  addi- 
tion to  it,  or,  before  sterilizing,  a  few  drops  of  litmus  tinc- 
ture may  be  added,  just  enough  to  give  it  a  pale-blue  color. 
By  this  means  it  will  be  seen  that  different  organisms  bring 
about  different  reactions  in  the  medium:  some  producing 
alkalies,  which  cause  the  blue  color  to  be  intensified;  others 
producing  acids,  which  change  it  to  red;  while  others 
bring  about  neither  of  these  changes.  Similarly  litmus 
solution  is  often  added  to  gelatin  and  agar-agar  for  the 
same  purpose. 

Milk  may  also  be  employed  as  a  solid  culture  medium 
by  the  addition  to  it  of  gelatin  or  agar-agar  in  the  propor- 
tions given  for  the  preparation  of  ordinary  nutrient  gelatin 
or  agar-agar.  It  has,  however,  in  this  form  the  disadvan- 
tage of  not  being  transparent,  and  can  therefore  best  be 
used  for  the  study  of  those  organisms  which  grow  upon  the 
surface  of  the  medium  without  causing  liquefaction. 

Nutrient  gelatin  and  agar-agar  can  also  be  prepared  from 
neutral  milk-whey,  obtained  from  milk  after  precipitation 
of  the  casein. 


1  For  some  time  past  we  have  been  using  what  is  technically  known 
as  "separator  milk" — i.  e.,  the  fluid  left  after  milk  has  been  deprived  of 
its  fat  (cream)  by  centrifugal  force. 


140  BACTERIOLOGY 

Litmus-whey  Milk. — An  important  differential  medium 
is  milk-whey  to  which  litmus  tincture  has  been  added. 
A  number  of  methods  for  its  preparation  are  in  use,  but  the 
one  employed  by  Durham  seems  to  be  the  most  satisfactory. 
Briefly  it  is  as  follows:  fresh  milk,  free  from  antiseptic 
adulterations,  is  gently  warmed  and  clotted  with  essence 
of  rennet.  The  whey  is  strained  off  and  the  clot  hung  up 
to  drain  in  a  piece  of  muslin.  The  whey,  which  is  somewhat 
turbid  and  yellow,  is  then  cautiously  neutralized  with  a  4 
per  cent,  citric  acid  solution,  neutral  litmus  solution  being 
used  as  the  indicator.  It  is  then  heated  upon  a  water-bath 
to  100°  C.  for  about  half  an  hour;  thereby  nearly  the  whole 
of  the  proteid  is  coagulated.  It  is  then  filtered  clear  and 
neutral  litmus  solution  is  added  until  it  is  of  a  distinct  purple 
color.  If  the  filtered  whey  is  cloudy,  let  it  stand  in  a  cold 
place  for  a  day  or  two  and  decant  off  the  clear  supernatant 
portion  or  pass  it  through  a  Berkefeld  filter.  The  whey  should 
never  be  heated  above  100°  C.  or  neutralized  with  mineral 
acids,  otherwise  there  is  a  danger  of  so  modifying  the  milk- 
sugar  present  as  seriously  to  impair  the  usefulness  of  the 
medium.  When  properly  prepared,  the  medium  is  free  from 
proteid,  and  contains  only  water,  lactose,  the  salts  of  the 
milk,  and  a  small  quantity  of  a  body  suggestive  of  dextrose 
or  galactose.  The  medium  is  of  great  utility  in  detecting 
the  power  of  bacteria  to  cause  acid  fermentation  in  a  non- 
proteid  medium  containing  a  fermentable  sugar;  and  for 
observing  the  variations  of  this  power  in  closely  allied  though 
not  identical  species. 

Dunham's  Peptone  Solution. — The  hiedium  known  as 
Dunham's  solution  is  prepared  according  to  the  following 
formula : 

Dried  peptone 1.0  part 

Sodium  chloride        .      .      .      .      .      .      .      .      .         0.5  part 

Distilled  water 100.0  parts 


DUNHAM'S  PEPTONE  SOLUTION  141 

It  is  usually  of  a  neutral  or  slightly  alkaline  reaction 
and  neutralization  is  not,  therefore,  necessary.  It  is  filtered, 
decanted  into  tubes  or  flasks,  and  sterilized  in  the  steam 
sterilizer  in  the  ordinary  way.  The  most  common  use  to 
which  this  solution  is  put  is  in  determining  if  the  organism 
under  consideration  possesses  the  property  of  producing 
indol  as  one  of  its  metabolic  products.  It  is  essential  for 
accuracy  that  the  preparation  of  dried  peptone  employed 
should  be  as  nearly  chemically  pure  as  is  possible,  and  indeed 
the  other  ingredients  should  be  correspondingly  free  from 
impurities.  Gorini1  calls  attention  to  the  fact  that  impurities 
in  the  peptone,  particularly  the, presence  of  carbohydrates, 
so  interfere  with  the  production  of  indol  by  certain  bacteria 
that  otherwise  produce  it,  that  it  is  ofttimes  impossible, 
under  such  circumstances,  to  obtain  the  characteristic 
color-reaction  of  this  body,  and  where  it  is  obtained  it  is 
always  after  a  much  longer  time  than  is  the  case  where  pep- 
tone free  from  these  substances  has  been  used. 

Peckham  has  also  demonstrated  that  where  bacteria 
have  the  property  of  forming  indol  and  also  of  fermenting 
carbohydrates,  their  proteolytic  function,  as  evidenced  by 
the  appearance  of  indol  as  a  product  of  metabolism,  may  be 
completely  suppressed  by  the  addition  of  such  fermentable 
carbohydrates  as  glucose,  saccharose,  and  lactose  to  the 
proteid  solution  in  which  they  are  developing.2 

Gorini  suggests  the  advisability  of  testing  the  purity  of 
all  peptone  preparations  before  using  them,  by  means  of 
the  reaction  that  they  exhibit  with  Fehling's  alkaline  copper 
solution.  Under  the  influence  of  this  reagent  pure  peptoen 
in  solution  gives  a  violet  color  (the  biuret  reaction),  which 

1  Centralblatt  fur  Bakteriologie  und  Parasitenkunde,  1893,  vol.  xiii,  p.  790. 

2  See  Journal  of  Experimental  Medicine,  1897,  vol.  ii,  p.  559. 


142  BACTERIOLOGY 

remains  permanent  even  after  boiling  for  five  minutes.  If, 
instead  of  a  violet  color,  there  appears  a  red  or  reddish- 
yellow  precipitate,  the  peptone  should  be  discarded,  as  in 
his  experience  no  indol  is  produced  from  peptone  giving 
this  reaction.  Both  the  peptone  solution  and  that  of  the 
copper  (particularly  the  latter)  should  be  relatively  dilute 
in  order  for  the  reaction  to  be  successful. 

Lactose  Litmus-agar,  or  Litmus-gelatin  of  Wurtz. — A 
medium  of  much  use  in  the  differentiation  of  bacteria  is 
that  recommended  by  Wurtz,  consisting  of  slightly  alkaline 
nutrient  agar-agar,  to  which  from  2  to  3  per  cent,  of  lactose 
and  sufficient  litmus  tincture  to  give  it  a  pale-blue  color  have 
been  added.  Bacteria  capable  of  causing  fermentation 
of  lactose  when  grown  on  this  medium  develop  into  colonies 
of  a  pale-pink  color  and  cause,  likewise,  a  reddening  of  the 
surrounding  medium,  owing  to  the  production  of  acid  as 
a  result  of  their  action  upon  the  lactose ;  while  other  bacteria, 
incapable  of  such  fermentative  activities,  grow  as  pale-blue 
colonies  and  cause  no  reddening  of  the  surrounding  medium. 
It  is  especially  useful  in  the  differentiation  of  the  bacillus 
of  typhoid  fever,  which  does  not  possess  the  property  of 
bringing  about  fermentation  of  lactose,  from  other  organ- 
isms that  simulate  it  in  many  other  respects,  but  which  do 
possess  this  property. 

Its  preparation  is  as  follows:  to  nutrient  agar-agar  or 
gelatin,  the  alkalinity  of  which  is  such  that  1  c.c.  will  require 
0.1  c.c.  of  a  1  : 20  normal  sulphuric-acid  solution  to  neu- 
tralize it,  lactose  is  added  in  the  proportion  of  2  or  3  per 
cent.;  it  is  then  decanted  into  test-tubes  and  sterilized  in 
the  usual  way.  When  sterilization  is  complete  enough 
sterilized  litmus  tincture  should  be  added  to  each  tube  to 
give  a  decided,  though  not  very  intense,  blue  color.  This 


SERUM  WATER  MEDIUM  OF  HISS  143 

must  be  done  carefully,  to  avoid  contamination  of  the  tubes 
during  manipulation.  It  is  better  not  to  add  the  litmus 
tincture  before  sterilizing  the  tubes,  as  its  color-character- 
istics are  altered  by  contact  with  organic  matters  under  the 
influence  of  heat.  This  medium  is  used  for  both  test-tube 
and  plate  cultivation,  just  as  is  ordinary  agar-agar  and 
gelatin. 

Lbffler's  Blood-serum  Mixture. — Loffler's  blood-serum  mix- 
ture consists  of  1  part  of  neutral  meat-infusion  bouillon, 
containing  1  per  cent,  of  grape  sugar,  and  3  parts  of  blood 
serum.  This  mixture  is  placed  in  test-tubes,  sterilized,  and 
solidified  in  exactly  the  way  given  for  blood  serum.  It 
requires  for  its  solidification  a  somewhat  higher  tempera- 
ture and  a  longer  exposure  to  this  temperature  than  does 
blood  serum  to  which  no  bouillon  has  been  added.  (See 
also  the  Councilman-Mallory  method.) 

The  Serum  Water  Medium  of  Hiss. — A  medium  which 
has  been  found  very  serviceable  in  the  differentiation 
between  closely  related  bacteria  is  prepared  by  mixing  1 
part  of  blood  serum  (either  horse  or  bovine)  and  3  parts  of 
distilled  water.  This  is  neutralized,  and  heated  in  a  water- 
bath  or  an  Arnold  steam  sterilizer  until  it  becomes  opales- 
cent. A  5  per  cent,  aqueous  solution  of  litmus  is  then 
added  in  the  proportion  of  1  per  cent.  Any  one  of  the 
carbohydrates,  as  dextrose,  lactose,  saccharose,  levulose, 
mannite,  etc.,  is  then  added  in  the  proportion  of  1  per  cent. 
The  finished  medium  is  then  placed  in  test-tubes.  The 
medium  must  be  sterilized  in  an  Arnold  steam  sterilizer,  and 
it  is  advisable  to  allow  the  sterilizer  to  remain  uncovered 
during  the  process  of  sterilization  to  avoid  excessive  heating 
of  the  medium. 

The  relative  degree  of  acidity  produced,  with  or  without 
coagulation,  with  or  without  gas-production,  and  with  or 


144  BACTERIOLOGY 

without  reduction  of  the  litmus,  in  a  series  of  tubes  of  this 
medium  containing  the  different  carbohydrates  serves  to 
differentiate  between  related  species  of  bacteria.  For 
instance,  the  colon  bacillus  produces  an  acid  reaction  with 
coagulation  and  gas-formation  with  some  of  the  carbohy- 
drates, while  the  typhoid  bacillus  produces  a  lower  degree 
of  acidity  with  coagulation,  but  without  gas-production. 
Similarly,  the  different  types  of  the  dysentery  bacillus  may 
be  differentiated  by  means  of  their  effects  on  the  different 
carbohydrates  in  this  medium. 

Guarniari's  Gelatin-agar  Mixture. — For  special  work, 
particularly  with  the  organism  of  pneumonia  (bacterium 
pneumonise)  the  gelatin-agar  mixture  recommended  by 
Guarniari  is  of  very  great  service.  It  should  be  exactly 
neutral  in  reaction,  and  should  possess  the  following  ingredi- 
ients : 

Meat  infusion 950  c.c. 

Sodium  chloride 5  grams 

Peptone    .     v'    .      .      .      .      .      .      .      .      .  25  to  30  grams 

Gelatin     .      ....••. .  40  to  60  grams 

Agar-agar 3  to    4  grams 

Water 50  c.c. 

The  agar-agar  should  be  completely  dissolved  separately 
in  about  100  c.c.  of  water  in  the  autoclave  while  the  other 
ingredients  are  being  prepared.  The  latter  should  be  filtered 
and  the  dissolved  agar-agar  added  to  the  filtrate. 

A  complete  list  of  the  special  media  would  be  too  volu- 
minous for  a  book  of  this  size.  For  their  description  the 
reader  is  referred  to  the  current  literature.  Those  that  have 
been  given  above  will  suffice  for  obtaining  a  clear  under- 
standing of  the  principles  of  the  subject.  In  the  chapters 
upon  the  Pathogenic  Bacteria  such  special  media  as  have 
proved  of  use  for  purposes  of  identification  and  differentiation 
are  described  in  detail. 


CHAPTER  VI. 

Preparation  of  the  Tubes,  Flasks,  etc.,  in  which  the  Media  are  to  be 
Preserved. 

WHILE  the  media  are  in  course  of  preparation  it  is  well 
to  get  the  test-tubes  and  flasks  ready  for  their  reception, 
and  it  is  essential  that  they  should  be  as  clean  as  it  is  pos- 
sible to  make  them.  For  this  purpose  it  is  advisable  that 
both  new  tubes  and  those  which  have  previously  been  used 
should  be  boiled  for  about  thirty  to  forty-five  minutes  in 
a  2  to  3  per  cent,  solution  of  common  soda ;  it  is  not  necessary 
to  be  exact  as  to  strength,  but  it  should  not  be  weaker  than 
this.  At  the  end  of  this  time  they  are  to  be  carefully  swabbed 
out  with  a  cylindrical  bristle  brush,  preferably  one  with  a 
reed  handle  (Fig.  21,  a),  as  those  with  wire  handles  are  apt 
to  break  through  the  bottoms  of  the  tubes,  though  Messrs. 
Lentz  &  Sons,  of  Philadelphia,  have  in  large  part  eliminated 
this  objection  from  the  wire-handle  brush  depicted  in  Fig. 
21,  6.  All  traces  of  adherent  material  should  be  carefully 
removed.  When  the  tubes  are  quite  clean  they  may  be 
rinsed  in  a  warm  solution  of  commercial  hydrochloric  acid 
of  the  strength  of  about  1  per  cent.  This  is  to  remove  the 
alkali.  They  are  then  to  be  thoroughly  rinsed  in  clear, 
running  water,  and  stood  top  down  until  the  water  has 
drained  from  them.  When  dry  they  are  to  be  plugged  with 
raw  cotton;  this  requires  a  little  practice  before  it  can  be 
properly  done.  The  cotton  should  be  introduced  into  the 
mouths  of  the  tubes  in  such  a  way  that  no  cracks  or  creases 
10  (145) 


146 


BACTERIOLOGY 


exist.  The  plug  should  fit  neither  too  tightly  nor  too  loosely, 
but  should  be  just  firmly  enough  in  position  to  sustain  the 
weight  of  the  tube  into  which  it  is  placed  when  held  up  by 
the  portion  which  projects  from  and  overhangs  the  mouth 
of  the  tube.  The  tubes  thus  plugged  are  now  to  be  placed 
upright  in  a  wire  basket  and  heated  for  one  hour  in  the  hot- 
air  sterilizer  at  a  temperature  of  about  150°  C.  A  very  good 
guide  for  this  process  of  sterilization  is  to  observe  the  tubes 
from  time  to  time,  and  as  soon  as  the  cotton  has  become  a 
very  light-brown  color,  not  deeper  than  a  dark-cream  tint, 
to  consider  sterilization  complete.  The  tubes  are  then 
removed  and  allowed  to  cool. 

FIG.  21 


Brushes  for  cleaning  test-tubes. 

The  cotton  used  for  this  purpose  should  be  the  ordinary 
cotton  batting  of.  the  shops,  and  not  absorbent  cotton; 
the  latter  becomes  too  tightly  packed,  and  is,  moreover, 
much  too  expensive  for  this  purpose. 

Care  should  be  taken  not  to  burn  the  cotton,  otherwise 
the  tubes  will  become  coated  with  a  dark-colored,  empyreu- 
matic,  oily  deposit,  which  necessitates  recleansing. 

Filling  the  Tubes. — When  the  tubes  are  cold  they  may  be 
filled.  This  is  best  accomplished  by  the  use  of  a  separating 
funnel,  such  as  is  shown  in  Fig.  22.  The  liquefied  medium 
is  poured  into  this  funnel,  which  has  been  carefully  washed, 


FILLING  THE  TUBES  147 

and  by  pressing  the  pinchcock  with  which  the  funnel  is 
provided  the  desired  amount  of  material  (5-10  c.c.)  may 
be  allowed  to  flow  into  the  tubes  held  under  its  opening. 
It  is  not  necessary  to  sterilize  the  funnel,,  for  the  medium 
is  to  be  subjected  to  this  process  as  soon  as  it  is  in  the  test- 
tubes. 

FIG.  22 


Funnel  for  filling  tubes  with  culture  media. 

Care  should  be  taken  that  none  of  the  medium  is  dropped 
upon  the  mouth  of  the  test-tube,  otherwise  the  cotton  plug 
becomes  adherent  to  it,  and  is  not  only  difficult  to  remove, 
but  presents  a  very  untidy  appearance  and  interferes  mate- 
rially with  the  manipulations. 

As  soon  as  the  tubes  have  been  filled  they  are  to  be  steril- 
ized either  in  the  steam  sterilizer  at  100°  C.  for  fifteen 


148  BACTERIOLOGY 

minutes  on  each  of  three  successive  days,  being  kept  during 
the  intervals  at  room  temperature,  or  they  may  be  sterilized 
by  a  single  exposure  of  fifteen  minutes  in  the  autoclave  to  a 
temperature  equivalent  to  steam  under  about  one  atmosphere 
of  pressure. 

When  sterilization  is  complete  and  the  medium  in  the 
tubes  is  still  liquid,  some  of  them  may  be  placed  in  a  slant- 
ing position,  at  an  angle  of  about  ten  degrees  with  the  sur- 
face on  which  they  rest,  and  the  medium  allowed  to  solidify 
in  this  position.  These  are  for  the  so-called  slant-cultures. 
The  remainder  may  solidify  in  the  erect  position;  these 
serve  for  making  plates. 


CHAPTER  VII. 

Technique  of  Isolating  Bacteria  in  Pure  Culture  by  the  Plate  and  the 
Tube  Method. 


PLATES. 

THE  plate  method  can  be  employed  with  both  agar-agar 
and  gelatin.  It  cannot  be  practised  with  blood  serum, 
because  the  serum  when  once  solidified  cannot  again  be 
liquefied. 

Plates  are  usually  referred  to  as  "a  set."  This  term 
implies  three  individual  plates,  each  representing  a  mixture 
of  organisms  in  a  higher  state  of  dilution.  The  first  plate 
is  known  usually  as  "the  original,"  or  "plate  No.  1,"  the 
first  dilution  from  this  as  "plate  No.  2,"  and  the  second  as 
"plate  No.  3." 

In  the  preparation  of  a  set  of  plates  the  following  are  the 
steps  to  be  observed: 

Three  tubes,  each  containing  from  7  to  9  c.c.  of  gelatin 
or  agar-agar,  are  placed  in  a  warm  water-bath  until  the 
medium  has  become  liquid.  If  agar-agar  is  employed,  this 
is  accomplished  at  the  boiling-point  of  water;  if  gelatin  is 
used,  a  much  lower  temperature  suffices  (35°-40°  C.).  When 
liquefaction  is  complete  the  temperature  of  the  water,  in 
the  case  of  agar-agar,  must  be  reduced  to  41°-42°  C.,  at 
which  temperature  the  agar-agar  remains  liquid,  and  the 
organisms  may  be  introduced  into  it  without  fear  of  de- 
stroying their  vitality.  The  medium  being  now  liquid  and 

(149) 


150  .  BACTERIOLOGY 

of  the  proper  temperature,  a  very  small  portion  of  the 
mixture  of  organisms  to  be  studied  is  taken  up  with  a  steril- 
ized platinum  wire  (Fig.  23,  a)  about  5  cm.  long,  twisted 
into  a  small  loop  at  one  end  and  fused  into  a  bit  of  glass 
rod,  which  serves  as  a  handle,  at  the  other  extremity.  This 
loop  is  one  of  the  most  useful  of  bacteriological  instruments, 
as  there  is  hardly  a  manipulation  into  which  it  does  not 
enter.  Under  no  circumstances  is  it  to  be  employed  without 
having  been  passed  through  a  gas-flame  until  quite  hot, 
for  the  purpose  of  sterilization.  One  should  form  a  habit 
of  never  taking  up  one  of  these  platinum-wire  needles,  as 

FIG.  23 
a 


6 
Looped  and  straight  platinum  wires  in  glass  handles. 

they  are  called,  for  they  are  curved  and  straight  (Fig.  23,  6) 
as  well  as  looped,  without  passing  it  through  a  flame;  and 
the  sooner  the  beginner  learns  to  do  this  as  a  reflex  action, 
the  sooner  does  he  eliminate  one  of  the  possible  sources  of 
error  in  his  work.  It  must  be  remembered,  though,  that  it 
should  not  be  used  when  hot,  otherwise  the  organisms  taken 
upon  it  will  be  killed  by  the  high  temperature;  after  steril- 
ization in  the  flame  one  waits  for  a  few  seconds  until  it 
is  cool  before  using. 

A  minute  portion  of  the  material  under  consideration  is 
transferred  with  the  sterilized  loop  into  tube  No.  1,  "the 
original,"  where  it  is  thoroughly  disintegrated  by  gently 


PLATES  151 

rubbing  it  against  the  sides  of  the  tube.  The  more  carefully 
this  is  done  the  more  uniform  will  be  the  distribution  of 
the  organisms  and  the  better  the  results.  The  loop  is  then 
again  sterilized  and  three  of  its  loopfuls  are  passed,  without 
touching  the  sides  of  the  tube,  from  ''the  original"  into  tube 
No.  2,  where  they  are  carefully  mixed.  Again  the  loop  is 
sterilized,  and  again  three  dips  are  made  from  tube  No.  2 
into  tube  No.  3.  This  completes  the  dilution.  The  loop  is 
now  sterilized  before  laying  it  aside. 

FIG.  24 


Levelling  tripod  with  glass  cooling  chamber  for  plates. 

During  this  manipulation,  which  must  be  done  quickly 
if  agar-agar  be  employed,  the  temperature  of  the  water  in 
the  bath  in  which  the  tubes  stand  should  never  be  lower  than 
39°  C.,  and  never  higher  than  43°  C.  If  it  falls  below  38° 
C.,  the  agar-agar  solidifies,  and  can  only  be  redissolved  at 
a  temperature  that  would  be  destructive  to  the  organisms 
which  may  have  been  introduced  into  the  tubes.  This  is 


152 


BACTERIOLOGY 


not  of  so  much  moment  with  gelatin,  since  it  may  readily  be 
redissolved  at  a  temperature  not  detrimental  to  the  organ- 
isms with  which  the  tubes  may  have  been  inoculated.  When 
completed  the  dilutions  are  poured  into  sterilized  Petri 
dishes  to  cool  and  solidify,  thereby  fixing  the  bacteria  so 
that  the  individuals  may  develop  into  their  characteristic 
colonies  and  be  so  separated  from  one  another  as  to  permit 
of  easy  isolation  in  pure  culture. 

FIG.  25 


Petri  double  dish,  now  generally  used  instead  of  plates. 

The  Petri  dish  (Fig.  25)  is  of  glass ;  round  in  form,  about 
8  or  9  cm.  in  diameter  and  1.5  to  2  cm.  deep,  with  a  loosely 
fitting  cover.  To  hasten  the  solidification  of  the  medium 

FIG.  26 


Metal  cooling  stage 

the  dishes  may  be  cooled  by  placing  them  upon  a  cold 
surface,  such  as  is  provided  by  the  glass  cooling  stage  (Fig. 


TUBE  METHODS  153 

24),  when  packed  with  ice,  or  on  the  metal  cooler,  shown  in 
Fig.  26,  through  which  cold  water  circulates.  The  plates 
are  labeled  to  correspond  with  their  respective  dilutions 
and  are  then  set  aside,  protected  from  dust  and  light  until 
colony  development  begins.  In  the  case  of  gelatin  the 
plates  must  not  be  maintained  at  a  temperature  higher 
than  that  of  an  ordinary  living  room,  about  20°  to  22°  C.  being 
the  most  favorable.  In  the  case  of  agar-agar  the  plates  may 
be  maintained  at  the  temperature  of  the  animal  body,  i.  e., 
between  37°  and  38°  C. 

TUBE  METHODS. 

Esmarch  Tubes. — A  useful  modification  of  the  plating 
method  just  described  is  that  suggested  by  von  Esmarch. 
It  insures  the  greatest  security  from  contamination  by 
extraneous  organisms  and  requires  the  least  amount  of 
apparatus.  It  differs  from  the  other  methods  thus:  the 
dilutions  having  been  prepared  in  tubes  contain  a  smaller 
amount  of  medium  than  usual — as  a  rule,  not  more  than 
5  to  6  c.c. — are,  instead  of  being  poured  upon  plates  or  into 
dishes,  spread  over  the  inner  surf  ace  of  the  tubes  containing 
them,  and,  without  removing  the  cotton  plugs,  solidified 
in  this  position.  The  tubes  then  present  a  thin  cylindrical 
lining  of  gelatin  or  agar-agar,  upon  which  the  colonies 
develop.  In  all  other  respects  the  conditions  for  the  growth 
of  the  organisms  are  the  same  as  in  flat  plates. 

The  solidification  of  the  media  on  the  inner  sides  of  the 
tubes  is  best  accomplished  by  rolling  them  upon  a  block 
of  ice  (Fig.  27),  after  the  plan  devised  by  Booker  in  1887  in 
the  Pathological  Laboratory  of  the  Johns  Hopkins  Univer- 
sity. In  this  method  a  small  block  of  ice  only  is  needed. 


154  BACTERIOLOGY 

It  is  levelled  and  held  in  position  by  being  placed  upon  a 
towel  in  a  dish.  A  horizontal  groove  is  melted  in  the  upper 
surface  of  the  ice  with  a  test-tube  of  hot  water.  The  tubes 
to  be  rolled  are  then  held  in  an  almost — not  quite — hori- 
zontal position  and  twisted  between  the  fingers  until  the 
sides  are  moistened  by  the  contents  to  within  about  1  cm. 
of  the  cotton  plug,  care  being  taken  that  the  gelatin  does 
not  touch  the  cotton,  otherwise  the  latter  becomes  adherent 
to  the  sides  of  the  tube  and  is  difficult  to  remove.  The  tube 

FIG.  27 


Demonstrating  Booker's  method  of  rolling  Esmarch  tubes  on  a  block  of  ice. 

is  then  placed  in  the  groove  in  the  ice  and  rolled  until  its 
contents  are  solid. 

There  is  an  erroneous  impression  that  Esmarch  tubes  are 
not  a  success  when  made  from  ordinary  nutrient  agar-agar 
because  of  the  tendency  of  this  medium  to  shrink  and  slip 
to  the  bottom  of  the  tube.  This  slipping  down  of  the  agar- 
agar  is  due  to  the  water,  which  is  squeezed  from  it  during 
solidification,  getting  between  the  medium  and  the  walls 
of  the  tube.  .This  can  easily  be  overcome  by  allowing  the 


TUBE  METHODS  155 

rolled  tubes  to  remain  in  a  nearly  horizontal  position  for 
twenty-four  hours  after  rolling  them,  the  mouth  of  the  tube 
being  about  1  cm.  higher  than  the  bottom.  During  this 
time  the  margin  of  the  agar-agar  nearest  the  cotton  plug 
dries  and  becomes  adherent  to  the  walls  of  the  tube,  while 
the  water  collects  at  the  most  dependent  point — i.  e.,  the 
bottom  of  the  tubes.  After  this  they  may  be  retained  in 
the  upright  position  without  danger  of  the  agar-agar  slipping 
down. 

In  both  the  plates  and  tubes,  if  the  dilutions  of  the  number 
of  organisms  have  been  properly  conducted,  the  results 
will  be  the  same.  The  original  plate  or  tube,  as  a  rule,  will 
be  of  no  use  because  of  the  great  number  of  colonies  in  it; 
plate  or  tube  No.  2  may  be  of  service;  but  plate  or  tube  No. 
3  will  usually  contain  the  organisms  in  such  small  numbers 
that  there  will  be  nothing  to  prevent  the  characteristic 
development  of  the  colonies  originating  from  them. 

For  reasons  of  economy  the  "original,"  tube  No.  1,  is 
sometimes  substituted  by  a  tube  containing  normal  salt- 
solution  (0.6  to  0.7  per  cent,  of  sodium  chloride  in  water), 
which  is  thrown  aside  as  soon  as  the  dilutions  are  completed, 
and  only  plates  or  tubes  Nos.  2  and  3  are  made. 

The  Serial  Tube  Method  of  Separation. — Another  method 
for  the  separation  of  bacteria  and  their  isolation  as  single 
colonies  consists  in  the  making  of  dilutions  upon  the  surface 
of  solid  media,  such  as  potato,  coagulated  blood  serum, 
agar-agar,  and  gelatin.  In  pursuance  of  this  method  one 
selects  a  number  of  tubes  containing  the  medium  set  in  a 
slanting  position.  With  a  platinum  needle  a  bit  of  the  sub- 
stance to  be  studied  is  smeared  upon  tube  No.  1;  without 
sterilizing  the  needle  it  is  passed  in  succession  over  the  surface 
of  the  medium  in  tubes  Nos.  2,  3,  4,  etc.  When  develop- 


156  BACTERIOLOGY 

ment  has  occurred  essentially  the  same  conditions  as  regards 
separation  of  the  colonies  will  be  found  as  when  plates  are 
poured.  If  a  slanted  medium  be  employed,  about  the  most 
dependent  angle  of  which  water  of  condensation  has  accu- 
mulated, as  blood  serum,  agar-agar,  and  potato,  the  dilu- 
tions may  be  made  in  this  fluid,  and  this  is  then  to  be  carefully 
smeared  over  the  solid  surface  of  the  medium.  The  tubes 
thus  treated  should  be  kept  in  an  upright  position  to  pre- 
vent the  fluid  flowing  over  the  surface.  When  sufficiently 
developed,  single  colonies  may  be  isolated  with  comparative 
ease  from  tubes  prepared  in  this  manner.  (See  also  method 
for  the  isolation  of  bacillus  diphtherise  on  blood  serum.) 


CHAPTER  VIII. 

The    Incubating    Oven — The    Safety   Burner    Employed   in    Heating   the 
Incubator — Thermo-regulator — Gas-pressure  Regulator. 


THE   INCUBATOR. 

WHEN  the  plates  have  been  made  it  must  be  borne  in 
mind  that  for  the  development  of  certain  forms  of  bacteria 
a  higher  temperature  is  necessary  than  for  the  growth  of 
others.  The  pathogenic  or  disease-producing  organisms  grow 
more  luxuriantly  at  the  temperature  of  the  human  body 
(37.5°  C.)  than  at  lower  temperatures;  whereas  for  the 
ordinary  saprophytic  forms  almost  any  temperature  be- 
tween 18°  and  37°  C.  is  suitable.  It  therefore  becomes 
necessary  to  provide  a  place  in  which  a  constant  tempera- 
ture favorable  to  the  growth  of  the  pathogenic  organisms 
can  be  maintained.  For  this  purpose  a  number  of  different 
forms  of  apparatus  have  been  devised.'  They  are  all  based 
upon  the  same  principles,  however,  and  a  general  description 
of  the  essential  points  involved  in  their  construction  will 
be  all  that  is  needed  here. 

The  apparatus  known  as  the  incubator,  or  brooding-oven, 
is  a  copper  chamber  (Fig.  28)  with  double  walls,  the  space 
between  which  is  filled  with  water.  The  incubating-chamber 
has  a  closely-fitting  double  door,  inside  of  which  is  a  door 
of  glass  through  which  the  contents  of  the  chamber  may  be 
inspected  without  actually  opening  it.  The  whole  apparatus 
is  encased  in  either  asbestos  boards  or  thick  felt,  to  prevent 
radiation  of  heat  and  consequent  fluctuations  in  teinpera- 

(157) 


158 


BACTERIOLOGY 


ture.  In  the  top  of  the  chamber  is  a  small  opening  through 
which  a  thermometer  projects  into  its  interior.  At  either 
corner,  leading  into  the  space  containing  the  water,  are 


FIG.  28 


Incubator  used  in  bacteriological  work. 

other  openings  for  the  reception  of  another  thermometer 
and  a  thermo-regulator,  and  for  refilling  the  apparatus  as 
the  water  evaporates.  On  the  side  is  a  water-gauge  for- 
showing  the  level  of  the  water  between  the  walls.  The 


SAFETY  BURNER 


159 


object  of  the  water-chamber,  which  is  formed  by  the  double- 
wall  arrangement,  is  to  insure,  by  means  of  the  warmed 
water,  an  equable  temperature  in  all  parts  of  the  apparatus — 
at  the  top  as  well  as  at  the  sides,  back,  and  bottom;  the 
apparatus  should  be  kept  filled  with  water,  otherwise  the 
purpose  for  which  it  is  constructed  will  not  be  served. 
When  the  chamber  between  the  walls  is  filled  with  water 
heat  is  supplied  by  a  gas-flame  placed  beneath  it. 

FIG.  29 


Koch's  safety  burner. 

The  burner  employed  in  heating  the  incubator  was  orig- 
inally devised  by  Koch,  and  is  known  as  *'  Koch's  safety 
burner"  (Fig.  291).  It  is  a  Bunsen  burner  provided  with 
an  arrangement  for  automatically  turning  off  the  gas-supply 

1  There  are  now  many  modifications  of  the  original  form. 


160  BACTERIOLOGY 

and  thus  preventing  accidents  should  the  flame  become 
extinguished  at  a  time  when  no  one  is  near.  The  gas-cock 
by  which  the  gas  is  turned  on  and  off  is  provided  with  a 
long  arm  which  is  weighted,  and  which,  when  the  gas  is 
turned  on  and  burning,  rests  upon  an  arm  attached  to  the 
side  of  a  revolving,  horizontal  disk  that  is  connected  with 
the  free  ends  of  two  metal  spirals  which  are  fixed  by  their 
other  ends  in  opposite  directions  on  either  side  of  the  flame 
and  heated  by  it.  If  by  draughts  or  any  other  accident 
the  flame  becomes  extinguished,  the  metal  spirals  cool,  and 
in  cooling  contract,  twist  the  horizontal  disk  in  the  opposite 
direction,  and  by  thus  removing  the  support  allow  the 
weighted  arm  of  the  gas-cock  to  fall.  By  its  falling  the  gas- 
supply  is  turned  off. 

Thermo-regulators. — The  regulation  and  maintenance  of 
the  proper  temperature  within  the  incubator  are  accom- 
plished by  the  employment  of  an  automatic  thermo-regulator 
or  thermostat. 

The  common  form  of  thermo-regulator  used  for  this 
purpose  is  constructed  uj>on  principles  involving  the  expan- 
sion and  contraction  of  fluids  under  the  influence  of  heat 
and  cold.  By  means  of  this  expansion  and  contraction  the 
amount  of  gas  passing  from  the  source  of  supply  to  the  burner 
may  be  either  diminished  or  increased  as  the  temperature 
of  the  substance  in  which  the  regulator  is  placed  either  rises 
or  falls. 

The  simplest  form  of  thermo-regulator  which  serves  to 
illustrate  the  principles  is  seen  in  Fig.  30.  It  consists  of  a 
glass  cylinder,  e,  having  a  communicating  branch  tube  6, 
and  rubber  stopper  /,  through  which  projects  the  bent  tube 
a.  The  tube  a  is  ground  to  a  slanting  point  at  the  extremity 
which  projects  into  the  tube  e,  and  is  provided  a  short  dis- 


THERMO-REG  ULA  TORS 


161 


FIG.  30 


/ 


tance  above  this  point  with  a  capillary  opening,  g,  in  one 
of  its  sides. 

When  ready  for  use  the  cylinder  e  is  filled  with  mercury 
up  to  about  the  level  shown  in  the  figure.    It  is  then  allowed 
to  stand,  or  is  suspended,  in   the 
bath  the  temperature  of  which  it 
is  to  regulate.    The  rubber  tubing 
coming  from  the  gas-supply  is  at- 
tached to  the    outer   end  of  the 
glass  tube  a,  and  the  tube  going  to 
the    burner    is   slipped    over    the 
branch  tube  b.    The  gas  is  turned 
on   and    the   burner    lighted :  and 
placed  under  the  bath.      The  gas 
now  streams  through  the   tube  a 
into  the  cylinder  e  and  out  at  b 
to  the  burner;    but  as    the  tem- 
perature of    the    bath    rises    the 
mercury  contained  in  the  cylinder 
e,  under  the  influence  of  the  ele- 
vated temperature,    begins   to  ex- 
pand, and,    as  a    continuous   rise 
in  temperature  proceeds,  the  expan- 
sion of  the  mercury  accompanies 
it  and  gradually  closes  the  slanting 
opening  h  of  tube  a.    In  this  way 
the  supply  of  gas  becomes  dimin- 
ished and  the  rise  in  temperature  of  the  bath  will  be  less 
rapid,  until  finally  the  opening  at  h  will  be  closed  entirely, 
when  the  supply  of  gas  to  the  burner  will  now  be  limited 
to  that  passing  through  the  capillary  opening  g.    This  is 
not  sufficient  to  maintain  the  highest  temperature  reached, 
11 


Mercurial  thermo-regulator. 


162  BACTERIOLOGY 

and  as  cooling  begins  a  gradual  contraction  of  the  mercury 
occurs  until  there  is  again  an  outflow  of  gas  from  the  opening 
h,  when  the  temperature  again  rises.  This  contraction  and 
expansion  of  the  mercury  in  the  regulator  continues  until 
eventually  a  point  is  reached  at  which  its  position  in  the 
cylinder  e  allows  of  the  passage  of  just  enough  gas  from  the 
opening  h  to  maintain  a  constant  temperature  and,  there- 
fore, a  constant  degree  of  expansion  of  the  mercury  in  the 
tube  e.  This,  in  short,  is  the  principle  on  which  thermo- 
regulators  are  constructed;  but  it  must  be  borne  in  mind 
that  a  great  deal  of  detail  exists  in  the  construction  of  an 
accurate  instrument.  The  number  of  different  forms  of 
this  apparatus  is  comparatively  large,  and  each  form  has 
its  special  merits. 

The  value — that  is,  the  delicacy — of  the  thermo-regulator 
depends  upon  a  number  of  factors,  all  of  which  it  would  be 
useless  to  describe  in  a  book  of  this  kind;  but  in  general 
it  may  be  said  that  the  essential  points  to  be  observed  in 
selecting  a  thermo-regulator  depend  in  the  main  upon 
the  temperatures  at  which  it  is  to  be  used.  For  low  tem- 
peratures, regulators  containing  such  fluids  as  ether,  alcohol, 
and  calcium  chloride  solution,  which  expand  and  contract 
rapidly  and  regularly  under  slight  variations  in  temperature, 
are  commonly  employed;  whereas  for  temperatures  approach- 
ing the  boiling-point  of  water  mercury  is  most  frequently  used. 

Other  types  of  regulators  operate  on  the  principle  of  the 
unequal  expansion  of  different  metals.  Thus,  if  two  strips 
of  metal  having  different  coefficients  of  expansion  be  fixed 
together,  when  expansion  occurs,  it  obviously  will  not  be 
in  a  right  line,  but  rather  at  an  angle  to  such  line.  Con- 
sequently if  one  end  of  such  a  composite  rod  be  fixed  and 
the  other  left  free,  the  higher  the  temperature  the  greater 


THERMO-REG  ULA  TORS 


163 


will  be  the  deflection  of  the  free  end  of  the  rod  from  the 
right  line;  the  lower  the  temperature  the  less  of  such  deflec- 
tion. If  now  the  free  tip  of  the  rod  be  so  connected  with 
the  gas  supply  that  with  increase  of  temperature  the  supply 
is  decreased  and  with  fall  of  temperature  increased,  it  is 
plain  that  by  proper  adjustment  the  gas  opening  can  be 

FIG.  31 


Moitessier's  gas-pressure  regulator. 


brought  to  a  point  that  will  supply  just  the  amount  of  gas 
needed  to  maintain  an  approximately  constant  temperature. 
The  temperature  of  the  incubator  is  to  be  regulated, 
then,  by  the  use  of  some  such  form  of  apparatus  as  those 
just  described.  The  regulator  should  be  of  sufficient  deli- 
cacy to  prevent  a  fluctuation  of  more  than  0.2°  C.  in  the 
temperature  of  the  air  within  the  chamber  of  the  apparatus. 


164  BACTERIOLOGY 

Gas-pressure  Regulators. — A  gas-pressure  regulator  is  some- 
times intervened  between  the  gas-supply  and  the  thermo- 
regulator.  This  apparatus  has  for  its  object  the  maintenance 
of  a  constant  pressure  of  the  gas  going  to  the  thermo-regu- 
lator.  There  are  several  forms  of  regulator  in  use,  but  they 
do  not  accomplish  the  object  for  which  they  are  designed. 

The  instrument  most  commonly  employed,  the  apparatus 
of  Moitessier  (Fig.  31),  is  based  on  somewhat  the  same 
principles  as  the  large  regulators  seen  at  the  manufactories 
of  illuminating-gas.  Such  apparatus  act  very  well  when 
employed  on  the  large  scale,  as  one  sees  them  at  the  gas- 
works; but  when  applied  to  the  limited  and  sudden  fluctua- 
tions seen  in  the  gas  coming  from  an  ordinary  gas-cock 
are  practically  useless.  They  are  too  gross  in  their  con- 
struction, and  act  only  under  comparatively  great  and 
gradual  fluctuations  in  pressure.  If  a  good  form  of  thermo- 
regulator  be  employed,  there  is  no  necessity  for  the  use  of 
any  of  the  pressure-regulators  thus  far  introduced. 


CHAPTER  IX. 

The  Study  of  Colonies — Their  Naked-eye  Peculiarities  and  Their  Appear- 
ance Under  Different  Conditions — Differences  in  the  Structure  of 
Colonies  from  Different  Species  of  Bacteria — Stab-cultures — Slant- 
cultures. 

THE  plates  of  agar-agar  which  have  been  prepared  from 
a  mixture  of  organisms  and  have  been  placed  in  the  incuba- 
tor, and  those  of  gelatin  which  have  been  maintained  at 
the  ordinary  temperature  of  the  room,  are  usually  ready  for 
examination  after  from  twenty-four  to  forty-eight  hours. 
They  will  be  found  marked  here  and  there  by  small  points 
or  little  islands  of  more  or  less  opaque  appearance.  In  some 
instances  these  will  be  so  transparent  that  it  is  with  diffi- 
culty one  can  see  them  with  the  naked  eye.  Again,  they 
may  be  of  a  dense,  opaque  appearance;  at  one  time  sharply 
circumscribed  and  round,  again  irregular  in  their  outline; 
here  a  point  will  present  one  color,  there  perhaps  another. 
On  gelatin  some  of  the  points  will  be  seen  to  be  lying  on  the 
surface  of  the  medium,  others  will  have  sunk  into  little 
depressions,  while  at  still  other  points  the  clear  gelatin  will 
be  marked  by  more  or  less  saucer-shaped  pits  containing 
opaque  fluid; 

Place  the  plate  containing  these  points  upon  the  stage  of 
a  microscope  and  examine  them  with  a  low-power  objec- 
tive, and  again  differences  will  be  observed.  Some  of  these 
minute  points  will  be  finely  granular,  others  coarsely  so; 
some  will  present  a  radiated  appearance,  while  a  neighbor 
may  be  concentrically  arranged;  here  nothing  particularly 
characteristic  will  present,  there  the  point  may  resolve 
itself  into  a  mass  having  somewhat  the  appearance  of  a 

(  165) 


166  BACTERIOLOGY 

little  pellicle  of  raw  cotton.  All  these  differences,  and  many 
more,  aid  us  in  saying  that  these  objects  must  be  different 
in  their  constitution.  With  a  pointed  platinum  needle  take 
up  a  bit  of  one  of  these  small  islands,  prepare  it  for  micro- 
scopic examination  (see  chapter  on  Stained  Cover-slip 
Preparations),  and  examine  it  under  the  high-power  oil- 
immersion  objective,  with  access  of  the  greatest  amount  of 
light  afforded  by  the  illuminator  of  the  microscope.  The 
preparation  will  be  seen  to  be  made  up  entirely  of  bodies 
of  the  same  shape;  they  will  all  be  spheres,  or  ovals,  or 
rods,  but  not  a  mixture  of  these  forms,  if  proper  care  in 
the  manipulation  had  been  taken.  Examine  in  the  same 
way  a  neighboring  spot  which  possesses  different  naked-eye 
appearances,  and  often  it  will  be  found  to  consist  of  bodies 
of  an  entirely  different  appearance  from  those  seen  in  the 
first  preparation. 

These  spots  or  islands  on  the  surface  of  the  plates  are 
colonies  of  bacteria,  differing  severally,  not  only  in  their 
gross  appearances,  the  one  from  the  other,  but,  as  our  cover- 
slip  preparations  show,  in  the  morphological  characteristics 
of  the  individual  organisms  composing  them. 

If  from  one  of  these  colonies  a  second  set  of  plates  be 
prepared,  the  peculiarities  which  were  first  observed  in  it 
will  be  reproduced  in  all  of  the  new  colonies  which  develop; 
each  will  be  found  to  consist  of  the  same  organisms  as  the 
colony  from  which  the  plates  were  made.  In  other  words, 
these  peculiarities  are  constant  under  uniform  conditions. 

The  appearance  of  the  colonies  developing  from  all  organ- 
isms is  regulated  by  their  location  in  the  medium  in  which 
they  are  growing.  When  deep  down  in  the  medium  they  are 
usually  round,  oval,  or  lozenge-shaped;  whereas  when  on 
the  surface  of  the  gelatin  or  agar  they  may  take  quite  a 


TEST-TUBE,  STAB  AND  SMEAR  CULTURES      167 

different  form.  This  is  purely  a  mechanical  effect  due  to 
the  pressure  of,  or  resistance  offered  by,  the  medium  sur- 
rounding them,  and  is  always  to  be  borne  in  mind,  other- 
wise false  interpretations  may  be  made. 

Pure  Cultures. — If  from  one  of  these  small  colonies  a  bit 
be  taken  upon  the  point  of  a  sterilized  platinum  needle  and 
introduced  into  a  tube  of  sterilized  gelatin  or  agar-agar,  the 
growth  that  results  will  be  what  is  known  as  a  "pure  culture," 
the  condition  to  which  all  organisms  must  be  brought  before 
a  systematic  study  of  their  many  peculiarities  is  begun. 
Sometimes  several  series  of  plates  are  necessary  before  the 
organisms  can  be  obtained  pure,  but  by  patiently  following 
this  plan  the  results  will  ultimately  be  satisfactory. 

Test-tube  Cultures;  Stab  Cultures;  Smear  Cultures. — After 
separating  the  organisms  the  one  from  the  other  by  the 
plate  method  just  described,  they  must  be  isolated  from  the 
plates  as  pure  stab  or  smear  cultures. 

This  is  done  in  the  following  way :  decide  upon  the  colony 
from  which  the  pure  culture  is  to  be  made.  Select  preferably 
a  small  colony  and  one  as  widely  separated  from  other 
colonies  as  possible.  Sterilize  in  a  gas-flame  a  straight 
platinum-wire  needle.  The  glass  handle  of  the  needle  should 
be  drawn  through  the  flame  as  well  as  the  needle  itself, 
otherwise  contamination  from  this  source  may  occur.  When 
it  is  cool,  which  is  in  five  or  ten  seconds,  take  up  carefully 
a  portion  of  the  colony.  Guard  against  touching  anything 
but  the  colony.  If  during  manipulation  the  needle  touches 
anything  else  whatever  than  the  colony  from  which  the  cul- 
ture is  to  be  made,  it  must  be  sterilized  again.  This  holds 
not  only  for  the  time  before  touching  the  colony,  but  also 
during  its  passage  into  the  test-tube  from  the  colony; 
otherwise  there  is  no  guarantee  that  the  growth  resulting 


168  BACTERIOLOGY 

from  the  inoculation  of  this  bit  of  colony  into  a  fresh  sterile 
medium  will  be  pure. 

In  the  meantime  have  in  the  other  hand  a  test-tube  of 
sterile  medium:  gelatin,  agar-agar,  or  potato.  This  tube  is 
held  across  the  palm  of  the  hand  in  an  almost  horizontal 
position  with  its  mouth  pointing  out  between  the  thumb 
and  index-finger  and  its  contents  toward  the  body  of  the 
worker.  With  the  disengaged  fingers  of  the  other  hand 
holding  the  needle  the  cotton  plug  is  removed  from  the 
tube  by  a  twisting  motion  and  placed  between  the  index 
and  second  fingers  of  the  hand  holding  the  tube,  in  such  a 
way  that  the  portion  of  the  plug  which  fits  into  the  mouth 
of  the  test-tube  looks  toward  the  dorsal  surface  of  the  hand 
and  does  not  touch  any  portion  of  the  hand;  this  is  accom- 
plished by  placing  only  the  overhanging  portion  of  the  plug 
between  the  fingers.  The  needle  containing  the  bit  of 
colony  is  now  to  be  thrust  into  the  medium  in  the  tube  if 
a  stab  culture  is  desired,  or  rubbed  gently  over  its  surface 
if  a  smear  or  stroke  culture  is  to  be  made.  The  needle  is 
then  withdrawn,  the  cotton  plug  replaced,  and  the  needle 
sterilized  before  it  is  laid  down.  Neither  the  needle  nor  its 
handle  should  touch  the  inner  sides  of  the  test-tube  if  it 
can  be  avoided.  The  tube  is  then  labelled  and  set  aside  for 
observation.  The  growth  which  appears  in  the  tube  after 
twenty-four  to  thirty-six  hours  should  be  a  pure  culture  of 
the  organisms  of  which  the  colony  was  composed. 

Cultures  of  this  form  are  not  only  useful  as  a  means  of 
preserving  the  different  organisms  with  which  we  may  be 
working,  but  serve  also  to  bring  out  certain  characteristics 
of  different  organisms  when  grown  in  this  way. 

If  gelatin  be  employed  and  the  organism  which  has  been 
introduced  into  it  possesses  the  power  of  bringing  about 


TEST-TUBE,  STAB  AND  SMEAR  CULTURES      169 

liquefaction — i.  e.,  of  digesting  it — it  will  soon  be  discovered 
that  the  mode  of  liquefaction  differs  with  different  organ- 
isms and  is  practically  constant  for  the  same  organism. 

FIG.  32 


Series  of  stab  cultures  in  gelatin,  showing  modes  of  growth  of  different 
species  of  bacteria. 

Some  bacteria  cause  a  liquefaction  which  spreads  across  the 
whole  upper  surface  of  the  gelatin  and  continues  gradually 
downward;  with  others  it  occurs  in  a  funnel-shape,  the 
broad  end  of  the  funnel  being  uppermost  and  the  point  down- 


170  BACTERIOLOGY 

ward,  corresponding  to  the  track  of  the  needle;  at  times  a 
stocking-  or  sac-like  liquefaction  may  be  noticed.  (See 
Fig.  32.) 

NOTE. — Obtain  a  number  of-  organisms  from  different 
sources  in  pure  cultures  by  the  method  given.  Plant  them 
as  pure  cultures,  all  at  the  same  time,  in  gelatin — preferably 
gelatin  of  the  same  making — retain  them  under  the  same 
conditions  of  temperature,  and  sketch  the  finer  differences 
in  the  way  in  which  liquefaction  occurs. 

Select  from  your  collection  a  non-spore-bearing,  actively 
liquefying  species.  Cultivate  it  as  a  pure  culture  in  nutrient 
bouillon  for  three  days.  Then  heat  this  bouillon  culture  to 
68°  C.  on  a  water-bath  for  ten  minutes.  In  the  meantime 
prepare  several  tubes  containing  each  about  10  c.c.  of: 

Gelatin 7 . 00  grams 

Phenol 0 . 25  gram 

Water 100. 00  c.c. 

Let  the  carbolized  gelatin  in  one  tube  remain  solid,  and 
bring  that  in  another  to  a  liquid  state  by  gentle  heat.  On 
the  surface  of  the  gelatin  in  the  first  tube  place  0.5  c.c. 
of  the  heated  (and  cooled)  culture,  and  mark  on  the  side  of 
the  tube  the  point  of  contact  between  the  fluid  culture  and 
the  solid  gelatin.  To  the  tube  of  liquefied  gelatin  add  like- 
wise 0.5  c.c.  of  the  heated  culture,  mix  it  thoroughly  with 
the  gelatin,  and  place  the  tube  containing  the  mixture 
in  cold  water  until  the  mass  becomes  solid.  Set  both  tubes 
aside  at  a  temperature  not  above  20°  C.  Note  what  occurs 
at  the  end  of  an  hour,  by  next  day,  and  after  three  days. 
Alter  the  experiment  by  filtering  the  three-day-old  bouillon 
culture  through  a  porcelain-  or  a  Berkefeld  filter,  instead  of 
heating  it  as  directed  above.  Are  the  results  modified? 
How  do  you  interpret  these  results? 


CHAPTER  X. 

Methods  of  Staining — Cover-slip  Preparations — Impression  Cover-slip 
Preparations — Solutions  Employed — Preparation  and  Staining  of 
Cover-slips — Staining  Solutions — Special  Staining  Methods. 

A  COMPLETE  list  of  solutions  and  methods  that  are 
recommended  for  the  staining  of  bacteria  is  not  essential 
to  the  work  of  the  beginner,  so  that  only  those  which  are  of 
the  most  common  application  will  be  given  in  this  book. 
In  general,  it  suffices  to  say  that  bacteria  stain  best  with 
watery  solutions  of  the  basic  aniline  dyes,  and  of  these, 
fuchsin,  gentian-violet,  and  methylene-blue  are  those  most 
frequently  employed. 

In  practical  work  bacteria  are  either  dried  upon  cover- 
slips  and  then  stained,  or  stained  in  sections  of  tissues  in 
which  they  have  been  deposited  during  the  course  of  disease. 
In  both  processes  the  essential  point  to  be  borne  in  mind 
is  that  the  bacteria,  because  of  their  microscopic  dimen- 
sions, require  to  be  more  conspicuously  stained  than  the 
surrounding  materials  upon  the  cover-slips  or  in  the  sec- 
tions, otherwise  their  recognition  is  a  matter  of  the  greatest 
difficulty,  if  not  of  impossibility.  For  this  reason,  especially 
in  section-staining,  it  frequently  becomes  necessary  to 
decolorize  the  tissues  after  removing  them  from  the  staining- 
solutions,  in  order  to  render  the  bacteria  more  prominent, 
and  for  this  purpose  special  methods,  which  provide  for 
decolorization  of  the  tissues  without  robbing  the  bacteria 
of  their  color,  are  employed.  The  ordinary  method  of 
cover-slip  examination  of  bacteria,  constantly  in  use  in  these 
studies,  is  performed  in  the  following  way: 

(171) 


172  BACTERIOLOGY 

COVER-SLIP   PREPARATIONS. 

In  order  that  the  distribution  of  the  organisms  upon  the 
cover-slips  may  be  uniform  and  in  as  thin  a  layer  as  possible 
it  is  essential  that  the  slips  should  be  clean  and  free  from 
grease.  For  cleansing  the  slips  several  methods  may  be 
employed. 

The  simplest  plan  with  new  cover-slips  is  to  immerse 
them  for  a  few  hours  in  strong  nitric  acid,  after  which  they 
are  rinsed  in  water,  then  in  alcohol,  then  ether,  and,  finally, 
they  may  be  kept  in  alcohol  to  which  a  little  ammonia  has 
been  added.  When  about  to  be  used  they  should  be  wiped 
dry  with  a  clean  cotton  or  silk  handkerchief. 

A  method  commonly  employed  is  to  remove  all  coarse 
adherent  matter  from  slips  and  slides  by  allowing  them  to 
remain  for  a  time  in  strong  nitric  or  sulphuric  acid.  They 
are  removed  from  the  acid  after  several  days,  rinsed  in  water, 
and  treated  as  above.  Knauer  suggests  the  boiling  of  soiled 
cover-slips  and  slides  for  from  twenty  to  thirty  minutes  in 
a  10  per  cent,  watery  solution  of  lysol,  after  which  they  are 
to  be  rinsed  carefully  in  water  until  all  trace  of  the  lysol  has 
disappeared.  They  are  then  to  be  wiped  dry  with  a  clean 
handkerchief. 

Loffler's  method,  which  provides  for  the  complete  removal 
of  all  grease,  is  to  warm  the  cover-slips  in  concentrated 
sulphuric  acid  for  a  time  and  then  rinse  them  in  water, 
after  which  they  are  kept  in  a  mixture  of  equal  parts  of 
alcohol  and  ammonia.  They  are  to  be  dried  on  a  cloth 
from  which  all  fat  has  been  extracted. 

Steps  in  Making  the  Preparations. — Place  upon  the  center 
of  one  of  the  clean  dry  cover-slips  a  very  small  drop  of  water 
or  physiological  salt-solution.  With  a  platinum  needle, 


COVER-SLIP  PREPARATIONS  173 

which  has  been  sterilized  in  a  gas-flame  just  before  using  and 
allowed  to  cool,  take  up  a  very  small  portion  of  the  colony 
to  be  examined  and  mix  it  carefully  with  the  drop  on  the 
slip  until  there  exists  a  very  thin  homogeneous  film  over 
the  larger  part  of  the  surface.  This  is  to  be  dried  upon  the 
slip  by  either  allowing  it  to  remain  upon  the  table  in  the 
horizontal  position  under  a  cover,  to  protect  it  from  dust, 
or  by  holding  it  between  the  fingers  (not  with  forceps} ,  at  some 
distance  above  a  gas-flame,  until  it  is  quite  dry.  If  held 
with  the  forceps  over  the  flame  at  this  stage,  too  much  heat 
may  be  unconsciously  applied,  and  the  morphology  of  the 
organisms  in  the  preparation  distorted.  When  held  between 
the  fingers  with  the  thin  layer  of  bacteria  away  from  the  flame 
no  such  accident  is  likely  to  occur.  When  the  whole  pellicle 
is  completely  dried  the  slip  is  to  be  taken  up  with  forceps, 
and,  holding  the  side  upon  which  the  bacteria  are  deposited 
away  from  the  direct  action  of  the  flame,  it  is  to  be  passed 
through  the  flame  three  times,  about  a  second  being  allowed 
for  each  transit.  Unless  the  preliminary  drying  at  the  low 
temperature  has  been  complete,  the  preparation  will  be 
rendered  worthless  by  the  subsequent  "fixing"  at  the  higher 
temperature,  for  the  reason  that  the  protoplasm  of  bacteria 
when  moist  coagulates  at  these  temperatures,  and  in  doing 
so  the  normal  outline  of  the  cells  is  distorted.  If  carefully 
dried  before  fixing,  this  does  not  occur  and  the  morphology 
of  the  organism  remains  unchanged. 

A  better  plan  for  the  process  of  fixing  is  to  employ  a 
copper  plate  about  35  cm.  long  by  10  cm.  wide  by  0.3  cm. 
thick.  This  plate  is  laid  upon  an  iron  tripod  and  a  small 
gas-flame  is  placed  beneath  one  of  its  extremities.  By  this 
arrangement  one  can  get  a  graduated  temperature,  beginning 
at  the  part  of  the  plate  above  the  gas-flame  where  it  is 


174  BACTERIOLOGY 

hottest,  and  becoming  gradually  cooler  toward  the  other  end 
of  the  plate,  which  may  be  of  a  very  low  temperature.  By 
dropping  water  upon  the  plate,  beginning  at  the  hottest 
point  and  proceeding  toward  the  cooler  end,  it  is  easy  to 
determine  the  point  at  which  the  water  just  boils;  it  is  at  a 
little  below  this  point  that  the  cover-slips  are  to  be  placed, 
bacteria  side  up,  and  allowed  to  remain  about  ten  minutes, 
when  the  fixing  will  be  complete.  In  very  particular  com- 
parative studies  this  plan  is  to  be  preferred  to  the  process 
of  passing  the  cover-slips  through  a  flame,  as  the  organisms 
are  always  subjected  to  the  same  degree  of  heat,  and  the 
distortions  which  sometimes  occur  from  too  great  and 
irregular  application  of  high  temperatures  may  be  elimi- 
nated. The  fixing  consists  in  drying  or  coagulating  the 
gelatinous  envelope  surrounding  the  organisms,  by  which 
means  they  are  caused  to  adhere  to  the  surface  of  the  cover- 
slip.  It  is  sometimes  desirable  to  fix  the  preparations  with- 
out the  use  of  heat,  as  in  the  case  of  pus  or  other  exudates. 
In  this  event,  after  drying  the  thinly  spread  material  care- 
fully m  the  air,  the  cover-slip  on  which  it  is  placed  is  im- 
mersed in  a  mixture  of  equal  parts  of  absolute  alcohol  and 
ether  for  about  fifteen  minutes.  At  the  end  of  this  time  it 
may  be  removed  and  stained.  The  advantage  of  this  method 
is  that  there  is  less  distortion  and,  as  a  rule,  less  precipitation 
(or,  perhaps  better,  no  charring)  of  extraneous  matter. 

The  majority  of  bacteria  with  which  the  beginner  will 
have  to  deal  stain  readily  with  watery  solutions  of  any  of 
the  basic  aniline  dyes,  such,  for  instance,  as  fuchsin,  methyl- 
ene-blue,  or  gentian-violet. 

To  stain  the  fixed  cover-slip  preparation,  it  is  taken  by 
one  of  its  edges  between  forceps,  and  a  few  drops  of  a  watery 
solution  of  either  of  the  dyes  named  are  placed  upon  the 


COVER-SLIP  PREPARATIONS  175 

film  and  allowed  to  remain  twenty  to  thirty  seconds.  The 
slip  is  then  carefully  rinsed  in  water,  and  without  drying 
is  placed  bacteria  down  upon  a  slide;  the  excess  of  water  is 
taken  up  by  covering  it  with  blotting-paper  and  gently 
pressing  upon  it,  after  which  the  preparation  is  ready  for 
examination. 

Another  plan  sometimes  used  is  to  bring  the  slip  upon 
the  slide,  bacteria  down,  without  rinsing  off  the  staining- 
fluid;  the  excess  of  fluid  is  removed  with  blotting-paper  and 
the  preparation  is  ready  for  examination  with  the  micro- 
scope. This  method  is  satisfactory  and  time-saving,  but 
must  always  be  practiced  with  care.  The  staining-fluid 
should  always  be  filtered  before  using,  to  rid  it  of  insoluble 
particles  which  might  be  taken  for  bacteria. 

If  upon  examination  the  preparation  prove  of  particular 
interest,  so  that  it  is  desirable  to  preserve  it,  then  it  may  be 
mounted  permanently.  The  drop  of  immersion  oil  is  to 
be  removed  from  the  surface  of  the  slip  with  blotting-paper, 
and  the  slip  loosened,  or  rather  floated,  from  the  slide  by 
allowing  water  to  flow  around  its  edges.  It  is  then  taken 
up  with  forceps,  carefully  deprived  of  the  water  adhering  to 
it  by  means  of  blotting-paper,  and  allowed  to  dry.  When 
dry  it  is  mounted  in  xylol-Canada-balsam  by  placing  a 
small  drop  of  the  balsam  upon  the  surface  of  the  film,  and 
then  inverting  the  slip  upon  a  clean  glass  slide.  It  is  some- 
times desirable  to  have  the  balsam  harden  quickly,  and  a 
method  that  is  commonly  employed  to  induce  this  is  as 
follows :  the  slide,  held  by  one  of  its  ends  between  the  fingers, 
is  warmed  over  a  gas-flame  until  quite  hot;  a  drop  of  balsam 
is  then  placed  on  the  center  of  it,  and  it  is  again  warmed; 
the  cover-slip  is  then  placed  in  position,  and  when  the  bal- 
sam is  evenly  distributed  the  temperature  is  rapidly  reduced 


176  BACTERIOLOGY 

by  rubbing  the  bottom  of  the  slide  with  a  towel  wet  with 
cold  water.  Usually  the  preparation  is  firmly  fixed  after 
this  treatment;  a  little  practice  is  necessary,  however,  in 
order  not  to  overheat  and  crack  the  slide.  The  method  is 
applicable  only  to  cover-slip  preparations,  and  cannot  be 
safely  used  with  tissues. 

Impression  Cover-slip  Preparations. — Impression  prepara- 
tions differ  from  ordinary  cover-slip  preparations  in  only 
one  respect:  they  present  an  impression  of  the  organisms 
as  they  were  arranged  in  the  colony  from  which  the  prep- 
aration is  made.  They  are  made  by  gently  covering  the 
colony  with  a  thin,  clean  cover-slip,  lightly  pressing  upon  it, 
and,  without  moving  the  slip  laterally,  lifting  it  by  one  of 
its  edges.  The  organisms  adhere  to  the  slip  in  the  same 
relation  to  one  another  that  they  had  in  the  colony.  The 
subsequent  steps  of  drying,  fixing,  staining,  and  mounting 
are  the  same  as  those  just  given  for  ordinary  cover-slip 
preparations. 

By  this  method  constancies  in  the  arrangement  and  group- 
ing of  the  individuals  in  a  colony  can  often  be  made  out. 
Some  will  always  appear  irregularly  massed,  others  show 
growth  in  parallel  bundles,  while  others,  again,  will  be  seen 
as  lorjg,  twisted  threads. 

NOTE. — From  a  colony  of  bacillus  subtilis  make  a  cover- 
slip  preparation  in  the  ordinary  way;  now  make  an  impres- 
sion cover-slip  preparation  of  another  colony  of  the  same 
organism.  Compare  the  results. 

ORDINARY   STAINING  SOLUTIONS. 

The  solutions  commonly  employed  in  staining  cover-slip 
preparations  are,  as  has  been  stated,  watery  solutions  of 


ORDINARY  STAINING  SOLUTIONS  177 

the  basic  aniline  dyes — fuchsin,  gentian-violet,  and  methyl- 
ene-blue.  These  solutions '  may  be  made  either  by  directly 
dissolving  the  dyes  in  substance  in  water  until  the  proper 
degree  of  concentration  has  been  reached,  or  by  using  con- 
centrated watery  or  alcoholic  solutions  of  the  dyes  which 
may  be  kept  on  hand  as  stock.  The  latter  method  is  the 
one  commonly  practised. 

The  solutions  of  the  colors  which  are  in  constant  use  in 
staining  are  prepared  as  follows : 

Prepare  as  stock,  saturated  alcoholic  or  watery  solutions 
of  fuchsin,  gentian-violet,  and  methylene-blue.  These 
solutions  are  best  made  by  pouring  into  clean  bottles  enough 
of  the  dyes  in  substance  to  fill  them  to  about  one-fourth  of 
their  capacity.  Each  bottle  should  then  be  filled  with 
alcohol  or  with  water,  tightly  corked,  well  shaken,  and 
allowed  to  stand  for  twenty-four  hours.  If  by  then  all  the 
staining  material  has  been  dissolved,  more  should  be  added, 
the  bottle  being  again  shaken  and  allowed  to  stand  for 
another  twenty-four  hours;  this  must  be  repeated  until 
a  permanent  sediment  of  undissolved  coloring  matter  is 
seen  upon  the  bottom  of  the  bottle.  The  bottles  are  then 
to  be  labelled  "saturated  alcoholic"  or  "watery"  solution 
of  fuchsin,  gentian-violet,  or  methylene-blue,  as  the  case 
may  be.  These  alcoholic  solutions  are  not  directly  employed 
for  staining-purposes. 

The  solutions  with  which  staining  is  accomplished  are 
made  from  the  stock  solutions  by  adding  5  c.c.  of  the  latter 
to  95  c.c.  of  distilled  water.  These  represent  the  staining 
solutions  in  every-day  use.  They  may  be  kept  in  bottles 
supplied  with  stoppers  and  pipettes  (Fig.  33),  and  when 
used  are  dropped  upon  the  preparation  to  be  stained. 

For  certain  bacteria  which  stain  only  imperfectly  with 
12 


178  BACTERIOLOGY 

these  simple  solutions  it  is  necessary  to  employ  agents 
that  will  increase  the  penetrating  action  of  the  dyes.  Ex- 
perience has  taught  us  that  this  can  be  accomplished  by  the 
addition  to  the  solutions  of  small  quantities  of  alkaline 
substances,  or  by  dissolving  the  staining  materials  in  strong 
watery  solutions  of  either  aniline  or  carbolic  acid,  instead 
of  water— in  other  words,  by  employing  special  solvents 
and  mordants  with  the  stains. 

FIG.  33 


Rack  of  bottles  for  staining  solutions. 

Of  the  solutions  thus  prepared  which  may  always  be 
employed  upon  bacteria  that  show  a  tendency  to  stain 
imperfectly,  there  are  three  in  common  use — Loffler's 
alkaline  methylene-blue  solution;  the  Koch-Ehrlich  ani- 
line-water solution  of  either  fuchsin,  gentian-violet,  or 
methylene-blue;  and  Ziehl's  solution  of  fuchsin  in  carbolic 
acid.  These  solutions  are  as  follows: 

Lqffler's  alkaline  methylene-blue  solution: 

Concentrated  alcoholic  solution  of  methylene-blue  30  c.c. 

Caustic  potash  in  1 : 10,000  solution 100  c.c. 

Koch-Ehrlich  aniline  water  solution.  To  about  100  c.c. 
of  distilled  water  aniline  oil  is  slowly  added,  a  few  drops 


ORDINARY  STAINING  SOLUTIONS  179 

at  the  time,  until  the  solution  has  an  opaque  appearance, 
the  vessel  containing  the  solution  being  thoroughly  shaken 
after  each  addition.  It  is  then  filtered  through  moistened 
filter-paper  until  the  filtrate  is  clear.  To  100  c.c.  of  the 
clear  filtrate  add  10  c.c.  of  absolute  alcohol  and  11  c.c.  of 
the  concentrated  alcoholic  solution  of  either  fuchsin,  methyl- 
ene-blue,  or  gentian-violet,  preferably  fuchsin  or  gentian- 
violet. 

Ziehl's  carbol-fuchsin  solution: 

Distilled  water 100  c.c. 

Carbolic  acid  (crystallized)       ......  5  grams 

Alcohol 10  c.c. 

Fuchsin  in  substance 1  gram 

Or  it  may  be  prepared  by  adding  to  a  5  per  cent,  watery 
solution  of  carbolic  acid  the  saturated  alcoholic  solution  of 
fuchsin  until  a  metallic  luster  appears  on  the  surface  of  the 
fluid. 

The  Koch-Ehrlich  solution  decomposes  after  a  time,  so 
that  it  is  better  to  prepare  it  fresh  in  small  quantities  when 
needed  than  to  employ  old  solutions.  Solutions  older  than 
fourteen  days  should  not  be  used. 

The  three  solutions  just  given  may  be  used  for  cover- 
glass  preparations  in  the  ordinary  way. 

In  some  manipulations  it  becomes  necessary  to  stain  the 
bacteria  very  intensely,  so  that  they  may  retain  their  color 
when  exposed  to  the  action  of  decolorizing  agents.  These 
methods  are  usually  employed  when  it  is  desirable  to  deprive 
surrounding  objects  or  tissues  of  their  color,  in  order  that 
the  stained  bacteria  may  stand  out  in  greater  contrast.  It 
is  in  these  cases  that  the  staining-solution  with  which  the 
bacteria  are  being  treated  is  to  be  warmed,  and  in  some 
cases  boiled,  so  as  further  to  increase  its  penetrating  action. 


180  BACTERIOLOGY 

When  so  treated,  certain  of  the  bacteria  will  retain  their 
color,  even  when  exposed  to  very  strong  decolorizers.  The 
tubercle  bacillus  is  distinguished  from  the  great  majority 
of  other  bacteria  by  the  tenacity  with  which  it  retains  the 
color  when  treated  in  this  way;  it  is  an  organism  difficult 
to  stain,  but  when  once  stained  is  equally  difficult  to  rob  of 
its  color. 

DECOLORIZING  SOLUTIONS. — As  regards  the  employment 
of  decolorizing  agents,  it  must  always  be  borne  in  mind  that 
objects  which  are  easily  stained  are  also  easily  decolorized, 
and  those  that  can  be  made  to  take  up  the  staining-material 
only  with  difficulty  are  also  very  difficult  to  rob  of  their 
color.  The  most  common  decolorizer  in  use  is  probably 
alcohol — not  absolute  alcohol,  but  alcohol  containing  more 
or  less  of  water.  Water  alone  has  this  property,  but  in  a 
much  less  degree  than  dilute  alcohol.  On  the  other  hand, 
a  much  more  energetic  decolorization  than  that  possessed 
by  either  alone  can  be  obtained  by  alternate  exposures  to 
alcohol  and  water.  More  energetic  in  their  decolorizing 
action  than  either  water  or  alcohol  are  solutions  of  the  acids. 
They  appear,  particularly  when  they  are  alcoholic  solutions, 
to  diffuse  rapidly  into  tissues  and  bacteria  and  very  quickly 
extract  the  staining  materials  which  have  been  deposited 
there.  For  this  reason  these  solutions  should  be  employed 
with  much  care. 

Very  dilute  acetic  acid  robs  tissues  and  bacteria  of  their 
stain  with  remarkable  activity;  still  more  energetic  are 
solutions  of  the  mineral  acids,  and  particularly,  as  has  been 
said,  when  this  action  is  accompanied  by  the  decolorizing 
properties  of  alcohol. 

The  acid  solutions  commonly  employed  are: 

Acetic  acid  in  from  0.1  to  5  per  cent,  watery  solution. 


STAINING  OF  TUBERCLE  BACILLI  181 

Nitric  acid  in  from  20  to  30  per  cent,  watery  solution. 

Sulphuric  acid  in  from  5  to  10  per  cent,  solution  in  water. 

Hydrochloric  acid  in  from  1  to  3  per  cent,  solution  in 
alcohol. 

NOTE. — For  details  as  to  the  technique  of  hardening  and 
cutting  sections  and  staining  bacteria  in  tissues,  the  student 
is  referred  to  Mallory  and  Wright's  Pathological  Technique. 

Method  of  Staining  the  Tubercle  Bacillus. — Select  from  the 
sputum  of  a  tuberculous  subject  one  of  the  small,  white, 
cheesy  masses  which  it  is  seen  to  contain.  Spread  this 
upon  a  cover-slip,  dry  and  fix  it  in  the  usual  way.  The  slip 
is  now  to  be  taken  by  its  edge  with  forceps  and  the  film 
covered  with  a  few  drops  of  either  the  solution  of  Koch- 
Ehrlich  or  that  of  Ziehl.  It  is  then  held  over  a  gas-flame, 
at  first  some  distance  away,  gradually  being  brought  nearer 
until  the  fluid  begins  to  boil.  After  it  has  bubbled  once  or 
twice  it  is  removed  from  the  flame,  the  excess  of  stain  washed 
away  in  a  stream  of  water,  then  immersed  in  a  30  per  cent, 
solution  of  nitric  acid  in  water,  and  allowed  to  remain  until 
all  color  has  disappeared.  This  takes  longer  in  some  cases 
than  in  others.  One  can  always  determine  if  decolorization 
is  complete  by  washing  off  the  acid  in  a  stream  of  water. 
If  the  preparation  is  still  distinctly  colored,,  it  should  be 
immersed  again  in  the  acid;  if  of  only  a  very  faint  color, 
it  may  be  dipped  in  alcohol,  again  washed  in  water,  and 
stained  with  some  contrast-color.  If,  for  example,  the 
tubercle  bacilli  have  been  stained  with  fuchsin,  methylene- 
blue  forms  a  good  contrast-stain.  In  making  the  contrast- 
stain  the  steps  in  the  process  are  exactly  those  followed  in 
the  ordinary  staining  of  cover-slip  preparations  in  general: 
the  slip  containing  the  stained  tubercle  bacilli  is  carefully 
rinsed  in  water,  and  a  few  drops  of  the  methylene-blue 


182  BACTERIOLOGY 

solution  placed  upon  it  and  allowed  to  remain  for  thirty 
or  forty  seconds,  when  it  is  again  rinsed  in  water  and 
examined  microscopically.  For  this  purpose  of  observing 
the  difference  in  behavior  of  the  tubercle  bacilli  and  the 
other  organisms  present  in  the  preparation  toward  this 
method  of  staining,  it  is  well  to  examine  the  preparation 
microscopically  before  the  contrast-stain  is  made;  then 
give  it  the  contrast-color,  and  again  examine.  It  will  be 
seen  that  before  the  contrast-color  has  been  given  to  the 
preparation  the  tubercle  bacilli  are  the  only  stained  objects 
to  be  made  out,  and  the  preparation  appears  devoid  of 
other  organisms;  but  upon  examining  it  after  it  has  received 
the  contrast-color  a  great  many  other  organisms  will  appear; 
these  take  on  the  second  color  employed,  while  the  tubercle 
bacilli  retain  their  original  color.  Before  decolorization 
all  organisms  in  the  preparation  were  of  the  same  color, 
bnt  during  the  application  of  the  decolorizing  solution  all 
except  the  tubercle  bacilli  gave  up  their  color.  This  micro- 
chemical  characteristic,  together  with  other  reactions  to 
be  described,  serves  to  differentiate  the  tubercle  bacillus 
from  other  organisms  with  which  it  might  be  confounded. 
A  number  of  different  methods  have  been  suggested  for  the 
staining  of  tubercle  bacilli,  but  the  original  method  as 
employed  by  Koch  is  so  satisfactory  in  its  results  that  it 
is  not  advisable  to  substitute  others  for  it.  The  above  differs 
from  the  original  Koch-Ehrlich  method  for  the  staining  of 
tubercle  bacilli  in  sputum  only  in  the  occasional  employ- 
ment of  Ziehl's  carbol-fuchsin  solution  and  in  the  method 
of  heating  the  preparation  with  the  staining  fluid  upon  it. 

As  Nuttall  has  pointed  out,  however,  the  strong  acid 
decolorizer  used  in  this  method  can,  with  advantage,  be 
replaced  by  much  more  dilute  solutions,  as  a  number  of  the 


DECOLORIZING  SOLUTIONS  183 

bacilli  are  entirely  decolorized  by  the  too  energetic  action 
of  the  strong  acids.  He  recommends  the  following  method 
of  decolorization :  after  staining  the  slip  or  section  in  the 
usual  way,  pass  it  through  three  alcohols;  it  is  then  to  be 
washed  in  a  solution  composed  of 

Water ..150  c.c. 

Alcohol 50  c.c. 

Concentrated  sulphuric  acid 20  to  30  drops 

From  this  it  is  removed  to  water  and  carefully  rinsed. 
The  remaining  steps  in  the  process  are  the  same  as  those 
given  in  the  other  methods. 

GABBETT'S  METHOD  for  the  staining  of  tubercle  bacilli 
recommends  itself  because  of  its  simplicity  and  the  rapidity 
with  which  it  can  be  performed.  By  many  it  is  considered 
the  best  method  for  routine  employment.  It  consists  in 
staining  the  cover-slips,  prepared  in  the  manner  given,  for 
from  two  to  five  minutes  in  a  cold  carbol-fuchsin  solution, 
after  which  they  are  subjected  to  the  action  of  Gabbett's 
methylene-blue  sulphuric  acid  solution.  This  latter  con- 
sists of 

Sulphuric  acid  (strength  25  per  cent.)      .      .     100  c.c. 
Methylene-blue,  in  substance 1  to  2  grams 

The.  cover-slips  are  then  rinsed  in  water  and  are  ready 
for  examination.  The  tubercle  bacilli  will  be  stained  red 
by  the  fuchsin,  while  all  other  bacteria,  cell-nuclei,  etc., 
will  be  tinted  blue. 

Pappenheim's  Decolorizer  and  Counter  Stain. — As  with  the 
Gabbett  method,  the  cover-slips  are  stained  for  from  5  to 
10  minutes  in  cold  carbol-fuchsin.  They  are  then  rinsed 
in  water  and  kept,  until  they  are  of  a  pale  blue  color,  in  a 
decolorizing  and  counter-staining  fluid  made  as  follows: 


184  BACTERIOLOGY 

To  100  c.c.  of  a  saturated  alcoholic  solution  of  methylene 
blue  add  1  gram  of  rosolic  acid  and  20  c.c.  of  glycerine. 
The  bacilli  are  stained  red,  the  balance  of  the  field  blue. 

Gram's  Method. — Another  important  differential  method 
of  staining  which  is  very  commonly  employed  is  that  recom- 
mended by  Gram.  In  this  method  the  objects  are  treated 
with  an  aniline-water  solution  of  gentian-violet  made  after 
the  formula  of  Koch-Ehrlich.  After  remaining  in  this  for 
two  or  three  minutes  they  are  immersed  in  a  solution  com- 
posed of 

Iodine 1  gram 

Potassium  iodide      .      .      .      .      .      .      .      .      .  2  grams 

Distilled  water    .      .      .      .      .      .....      .        300  c.c. 

In  this  they  remain  for  about  five  minutes;  they  are  then 
transferred  to  95  per  cent,  alcohol  and  thoroughly  rinsed. 

This  method  is  particularly  useful  in  demonstrating  the 
capsule  which  is  seen  to  surround  some  bacteria,  especially 
micrococcus  lanceolatus  of  pneumonia. 

After  such  treatment  certain  species  of  bacteria  are  found 
to  be  of  a  very  dark  purple  color,  while  all  else  in  the  prepa- 
ration is  decolorized;  other  species  lose  their  color  entirely 
in  the  process.  Those  that  retain  the  dark  stain  are  com- 
monly denominated  as  "  Gram-positive"  while  those  that 
lose  their  color  are  known  as  "Gram-negative."  While  the 
majority  of  bacteria  are  either  definitely  positive  or  negative 
to  this  reaction,  there  are  a  few  species  that  are  indeter- 
minate in  this  particular,  that  is  to  say,  they  become  partly 
decolorized  and  one  cannot  say  certainly  that  they  are 
either  positive  or  negative.  Under  certain  conditions  of 
cultivation,  and  especially  under  conditions  favorable  to 
degenerative  changes,  some  species  that  are  normally 


SPECIAL  STAINING  185 

"Gram-positive"  may  in  part  or  wholly  lose  their  "Gram- 
positive"  properties. 

Two  theories,  one  chemical  the  other  physical,  have  been 
offered  in  explanation  of  the  mechanism  of  the  Gram  method 
of  staining.  In  the  chemical  theory  it  is  believed  that, 
through  the  intervention  of  the  iodine,  the  gentian-violet 
is  linked  inseparably  to  the  protoplasm  of  "Gram-positive" 
bacteria  and  is  not  so  linked  in  the  "Gram-negative"  species. 
The  physical  theory  assumes  differences  in  permeability 
of  either  the  bacterial  envelope  or  the  bacterial  protoplasm. 
In  those  species  that  are  highly  permeable  the  precipitation 
resulting  from  the  interaction  between  the  iodine  and  the 
gentian-violet  occurs  so  deeply  within  the  bacterial  structure 
that  it  is  not  readily  washed  out  by  the  final  alcohol  bath, 
this  would  be  the  case  with  the  "Gram-positive"  species; 
while  in  the  case  of  the  "Gram-negative"  species,  assumed 
to  be  less  permeable,  the  precipitation  is  upon  their  sur- 
faces and  is  readily  removed  by  the  final  rinsing  in  alcohol. 

Glacial  Acetic  Acid  Method. — Another  method  that  may 
be  employed  for  demonstrating  the  presence  of  the  cap- 
sule surrounding  certain  organisms  is  to  prepare  the 
cover-slips  in  the  ordinary  way,  then  cover  the  layer 
of  bacteria  upon  them  with  glacial  acetic  acid,  which 
is  instantly  poured  off  (not  washed  off  with  water),  and 
the  aniline-water  gentian-violet  solution  dropped  upon 
them;  this  is  allowed  to  remain  three  or  four  minutes,  is 
poured  off,  and  a  few  drops  more  are  added,  and  lastly  the 
slip  is  washed  in  a  solution  of  sodium  chloride  of  from  0.6 
to  0.7  per  cent,  in  strength;  but  at  times  it  must  be  stronger, 
occasionally  as  concentrated  as  1.5  to  2  per  cent.  The  reason 
for  this  is  that  if  the  slips  be  washed  in  water,  or  in  salt- 
solution  that  is  too  weak,  the  mucin  capsule  that  has  been 


186  BACTERIOLOGY 

coagulated  by  the  acetic  acid  is  redissolved  and  rendered 
invisible.  This  does  not  occur  when  the  salt-solution  is 
of  the  proper  strength — a  point  that  can  be  determined  only 
after  a  few  trials  with  solutions  of  different  strengths. 
(Welch.)  A  very  clear,  sharply  cut  picture  usually  follows 
this  method  of  procedure. 

Ribbert  also  recommends  for  the  staining  of  capsulated 
bacteria  the  momentary  immersion  of  the  cover-slips  in 
a  saturated  solution  of  dahlia  in  a  mixture  of  100  parts  of 
water,  50  parts  of  alcohol,  and  12 J  parts  of  glacial  acetic 
acid;  after  which  the  excess  of  color  is  removed  by  washing 
in  water. 

Staining  of  Spores. — We  have  learned  that  one  of  the 
points  by  which  spores  may  be  recognized  is  their  refusal 
to  take  up  staining  substances  when  applied  in  the  ordinary 
way.  They  may,  however,  be  stained  by  special  methods; 
of  these,  one  that  has  given  fairly  satisfactory  results  in 
our  hands  is  as  follows:  the  cover-slip  is  to  be  prepared  from 
the  material  containing  the  spores  in  the  ordinary  way, 
dried,  and  fixed.  It  is  then  to  be  held  by  its  edge  with 
forceps,  and  its  surface  covered  with  Loffler's  alkaline 
methylene-blue  solution.  It  is  then  held  over  the  Bunsen 
flame  until  the  fluid  boils;  it  is  then  removed,  and  after  a 
few  seconds  is  heated  again.  This  is  continued  for  about 
one  minute,  after  which  it  is  washed  in  water  and  then 
decolorized  in 

Alcohol  (80  per  cent.) 98  c.c. 

Nitric  acid 2  c.c. 

until  all  visible  blue  color  has  disappeared.  It  is  then  rinsed 
in  water  and  dipped  for  from  3  to  5  seconds  in 

Saturated  alcoholic  solution  of  eosin 10  c.c. 

Water  .  ....      90  c.c. 


FLAGELLAR  STAINING  187 

after  which  it  is  again  rinsed  in  water  and  finally  mounted 
for  examination.  If  the  decolorization  in  the  acid  alcohol 
be  not  carried  too  far,  the  preparation  will  show  the  spores 
stained  blue  and  the  bodies  of  the  cells  to  have  taken  on 
the  rose  color  characteristic  of  eosin. 

By  another  process  the  cover-slip  is  floated,  bacteria 
down,  upon  the  surface  of  freshly  prepared  Koch-Ehrlich 
solution  of  fuchsin  contained  in  a  watch-crystal.  This  is 
then  held  by  its  edge  with  forceps  and  moved  up  and  down 
over  a  small  Bunsen  flame  until  the  fluid  boils  gently.  This 
is  continued  for  2  or  3  minutes.  When  the  fluid  has  stood 
for  about  five  minutes  after  boiling  the  preparation  is  trans- 
ferred, without  washing  in  water,  to  a  second  watch-crystal 
containing  the  following  decolorizing  solution : 

Absolute  alcohol 100  c.c. 

Hydrochloric  acid 3  c.c. 

In  this  solution  it  is  placed,  bacteria  up,  and  the  vessel  is 
tilted  from  side  to  side  for  about  one  minute.  It  is  then 
removed,  washed  in  water,  and  stained  with  the  cold 
methylene-blue  solution.  The  spores  will  be  stained  red 
and  the  body  of  the  cells  blue. 

It  must  be  remembered  that  there  are  conspicuous  dif- 
ferences in  the  behavior  of  spores  of  different  bacteria  to 
staining  methods  and  of  the  spores  of  a  single  species  in 
different  stages  of  development.  Some  stain  readily  by  either 
of  the  methods  especially  devised  for  this  purpose,  while 
others  can  hardly  be  stained  at  all,  or  only  with  the  greatest 
difficulty,  by  any  of  the  known  processes;  some  stain 
readily  when  fully  developed,  but  with  difficulty  when 
only  partly  developed;  others  have  this  peculiarity  reversed. 

Lbffler's  Method  for  Staining  Flagella. — For  the  demon- 
stration of  the  locomotive  apparatus  possessed  by  motile 


188  BACTERIOLOGY 

bacteria  we  are  indebted  to  Loffler.  By  a  special  method 
of  staining,  in  which  the  use  of  mordants  played  the  essen- 
tial part,  he  has  shown  that  these  organisms  possess  very 
delicate,  hair-like  appendages,  by  the  lashing  movements 
of  which  they  propel  themselves  through  the  fluid  in  which, 
they  are  growing.  The  method  as  given  by  Loffler  is  as 
follows: 

It  is  essential  that  the  bacteria  be  evenly  and  not  too 
numerously  distributed  upon  the  cover-slip.  The  slips  must 
therefore  be  perfectly  clean.  (See  Loffler1  s  method  of  clean- 
ing cover-slips.)  Five  or  six  of  the  carefully  cleansed  cover- 
slips  are  to  be  placed  in  a  line  on  a  table,  and  on  the  center 
of  each  slip  a  very  small  drop  of  tap-water  is  placed.  From 
the  culture  to  be  examined  a  minute  portion  is  transferred 
to  the  first  slip  and  carefully  mixed  with  the  drop  of  water; 
from  this  mixture  a  small  portion  is  transferred  to  the 
second,  and  from  the  second  to  the  third  slip,  and  so  on, 
in  this  way  insuring  a  dilution  of  the  number  of  organisms 
present  in  the  preparations.  These  slips  are  then  dried  and 
fixed  in  the  ordinary  way.  They  are  next  to  be  warmed  in 
the  following  solution: 

Tannic  acid  solution  in  water  (20  acid,  80  water)  .  10  c.c. 
Cold  saturated  solution  of  ferrous  sulphate  ...  5  c.c. 
Saturated  watery  or  alcoholic  solution  of  fuchsin  .  1  c.c. 

This  solution  represents  the  mordant.  A  few  drops  of 
it  are  to  be  placed  upon  the  film  of  bacteria  on  the  cover- 
slip,  which  is  then  to  be  held  over  a  flame  until  the  solution 
begins  to  steam.  It  should  not  be  boiled.  After  steaming, 
the  mordant  is  washed  off  in  water  and  finally  in  alcohol. 
The  bacteria  are  then  to  be  stained  in  a  saturated  aniline- 
water-fuchsin  solution. 

There  are  several  points  and  slight  modifications  in  con- 


FLAGELLAR  STAINING  189 

nection  with  this  method  that  require  to  be  emphasized  in 
order  to  insure  success:  the  culture  to  be  employed  should 
be  young,  not  over  18-20  hours  old;  it  should  have  developed 
for  this  time  on  fresh  agar-agar  at  37°  to  38°  C. ;  the  mordant 
should  not  be  perfectly  fresh,  as  the  best  results  are  obtained 
from  the  use  of  old  solutions  that  have  stood  exposed  to  the 
air  and  that  have  been  filtered  just  before  using;  when 
placed  on  the  cover-slip  and  held  over  the  flame  never  heat 
the  mordant  to  the  boiling-point;  'indeed,  the  best  results  are 
obtained  when  the  preparation  is  held  high  above  the  flame 
and  removed  from  it  at  the  first  evidence  of  vaporization,  or, 
better  still,  a  little  before  this  point  is  reached.1 

Duck-wall's  Method2  is  a  modification  of  the  Loffler  method, 
and  the  results  obtained  thereby  are  very  satisfactory. 

Preparation  of  the  Staining  Agents. — The  fixing  agent  is 
mordant,  and  the  stain  is  carbol-gentian-violet  or,  prefer- 
ably, carbol-fuchsin. 

The  Mordant. 

Desiccated  tannic  acid 2  grams 

Cold  saturated  solution  ferrous  sulphate  (aqueous)  5  grams 

Distilled  water 12  c.c. 

Saturated  alcoholic  solution  of  fuchsin  ....  1  c.c. 

The  tannic  acid  is  dissolved  in  the  water  first  by  the 
application  of  gentle  heat,  then  the  ferrous  sulphate,  and 
then  the  alcoholic  solution  of  fuchsin  are  added.  To  these 
ingredients  it  is  advisable  to  add  from  0.5  to  1  c.c.  of  a 
1  per  cent,  sodium  hydroxide  solution.  The  best  grade  of 
filter-paper  is  used  for  filtering  the  mordant,  and  there 
should  be  left  a  heavy  precipitate.  After  filtering,  the  color 

1 1  am  indebted  to  Dr.  James  Homer  Wright,  Thomas  Scott  Fellow  in 
Hygiene,  1892-1893,  University  of  Pennsylvania,  for  some  of  the  suggestions 
in  connection  with  the  modification  of  this  method. 

2  The  Canner,  vol.  xx,  p.  23. 


190  BACTERIOLOGY 

of  this  mordant  should  be  of  a  reddish-brown  hue,  not  clear, 
but  somewhat  cloudy,  and  this  mordant  must  be  used  within 
five  hours  after  it  is  made.  After  that  time  it  loses  its  fixing 
power.  This  is  indicated  by  its  gradual  clarification  and 
darkened  color.  It  gives  the  best  results  when  strictly 
fresh,  and  accomplishes  its  work  in  a  much  shorter  time,  so 
that  very  little  if  any  heating  is  required  when  it  is  placed 
on  the  cover-glass  preparation. 

The  Stain. — To  prepare  the  dye  for  this  method  take 
about  1  gram  of  ordinary  granulated  fuchsin,  put  it  in  a 
bottle,  and  pour  over  it  about  25  c.c.  of  warm,  absolute 
alcohol.  Shake  vigorously  and  let  it  stand  for  several 
hours  before  using.  The  carbol-fuchsin  is  made  by  diluting 
the  saturated  alcoholic  solution  four  or  five  times  with  a 
5  per  cent,  solution  of  carbolic  acid.  Carbol-fuchsin  should 
be  freshly  made,  heated,  and  filtered  before  using. 

The  application  of  this  method  of  demonstrating  the 
flagella  varies  with  different  organisms  with  regard  to  the 
length  of  time  the  mordant  and  stain  are  allowed  to  act, 
and  the  amount  of  sodium  hydroxide  solution  used.  Usually, 
it  is  well  to  heat  the  mordant  on  the  cover-slip  to  steaming, 
and  allow  it  to  act  from  one-half  to  one  minute.  It  is  then 
washed  off  with  water  and  a  small  quantity  of  alcohol 
poured  over  the  surface  and  washed  off  instantly.  The 
water  on  the  cover-slip  is  now  absorbed  from  the  edge  of 
the  cover-slip  with  clean  filter-paper.  The  carbol-fuchsin 
stain  is  now  applied  and  heated  just  enough  to  generate  a 
thin  vapor.  The  stain  should  not  act  for  more  than  from 
one-half  to  one  minute.  The  cover-slip  is  now  dried,  then 
xylol  is  poured  over  the  surface,  the  excess  being  removed 
with  filter-paper.  The  cover-slip  is  now  mounted  in  xylol 
balsam. 


CHAPTER    XL 

Systematic  Study  of  an  Organism — Points  to  be  Considered  in  Determin- 
ing the  Morphologic  and  Biologic  Characters  of  a  Culture — Methods 
by  Which  the  Various  Biologic  and  Chemical  Characters  of  a  Culture 
may  be  Ascertained — Dark  Field  Illumination — Facts  Necessary  to 
Permit  the  Identification  of  an  Organism  as  a  Definite  Species. 

AFTER  isolating  an  organism  in  pure  culture  by  the  plate 
method,  considerable  work  is  necessary  in  order  to  estab- 
lish its  identity.  Small  portions  of  the  pure  culture  are 
taken  upon  the  point  of  a  sterile  platinum  wire  and  trans- 
planted into  the  various  culture  media.  These  sub-cultures 
of  the  organism  are  then  placed  under  suitable  conditions 
of  temperature  and  environment,  and  examined  from  day 
to  day  to  note  the  alterations  that  occur  in  the  different 
media.  In  the  systematic  study  of  an  organism  no  one 
character  can  be  relied  upon  to  the  exclusion  of  others. 
It  is  necessary  to  note  the  microscopic  appearance  of  the 
individual  organism  and  its  behavior  toward  different 
staining  solutions  and  other  reagents;  in  addition  it  is 
necessary  to  note  the  gross  appearance  of  the  culture  of 
the  different  media  as  shown  by  naked-eye  (macroscopic) 
examination  as  well  as  under  a  lens  of  low  magnifying 
power  (microscopic);  while  equal  importance  must  be  given 
to  the  chemical  alterations  produced  by  the  bacteria  in  the 
different  media,  and  the  influence  of  different  reagents, 
when  added  to  the  media,  to  show  the  presence  of  certain 
metabolic  products.  In  this  manner  the  entire  life  history 
of  an  organism,  outside  the  animal  body,  may  be  ascertained. 

(191) 


192  BACTERIOLOGY 

The  different  characters  of  an  organism  may  be  grouped 
as:  (a)  morphologic,  those  ascertained  by  examination  of 
the  individual  organism  under  a  lens  of  high  magnifying 
power;  (b)  biologic,  those  ascertained  by  macroscopic  and 
microscopic  study  of  the  gross  appearance  of  the  culture 
in  the  different  media;  (c)  biochemic,  the  alterations  pro- 
duced in  the  different  media  as  shown  by  direct  examination 
or  by  the  use  of  different  reagents;  and  (d)  pathogenic, 
the  effects  of  the  inoculation  of  the  culture  into  susceptible 
animals. 

SCHEME  or  STUDY. — Record  the  source  whence  the 
organism  was  derived.  Was  this  the  normal  habitat  of  the 
organism,  or  was  it  present  accidentally? 

MORPHOLOGIC   CHARACTERS. 

Note  the  shape,  size,  and  grouping  of  the  organism  as  it 
occurs  in  the  different  media.  Observe  the  nature  of  the 
ends  of  the  individual  organism.  Determine  the  presence 
or  absence  of  motility  in  very  young  cultures.  If  motility 
is  observed,  apply  one  of  the  special  methods  for  demon- 
strating flagella  to  note  their  relative  number  and  location 
and  do  not  be  discouraged  if  your  first  attempts  fail.  Stain 
your  cultures  by  means  of  the  different  staining  solutions, 
and  note  the  effect  of  each.  Do  the  organisms  stain  deeply 
and  uniformly,  or  are  they  stained  in  a  peculiar  manner? 
Apply  the  Gram  method  of  staining,  and  note  whether  or 
not  the  organisms  are  decolorized  by  the  alcohol.  Stain 
the  organisms  deeply  with  carbol-fuchsin  staining  solution, 
and  note  the  effect  of  different  decolorizing  agents;  and 
ascertain  whether  the  organisms  are  capable  of  resisting  the 
decolorizing  effects  of  dilute  acids.  Do  the  organisms  show 


BIOLOGIC  CHARACTERS  193 

the  presence  of  a  capsule  when  taken  from  the  blood  or 
tissues  of  an  animal,  or  when  taken  from  cultures  in  milk 
or  blood-serum  ?  Examine  cultures  that  are  several  days  old, 
and  note  whether  spores  are  being  formed.  Note  particularly 
the  position  of  the  spore  within  the  cell.  Is  the  spore  of 
smaller  or  greater  diameter  than  the  cell  in  which  it  is 
forming?  Examine  cultures  that  are  a  week  or  more  old, 
and  note  whether  the  organisms  have  undergone  any  definite 
alterations  in  form  (involution  forms),  or  whether  they 
present  evidences  of  fragmentation  or  granulation  of  their 
protoplasm  (degeneration  forms) . 

BIOLOGIC  CHARACTERS. 

Colony  Formation. — Observe  the  character  of  the  colonies 
formed  in  gelatin  and  agar-agar  plates.  Describe  a  typical 
surface  colony  and  a  typical  deep  colony,  both  as  to  their 
macroscopic  and  microscopic  appearance.  What  is  the 
relative  size  of  the  colonies  formed  on  each  of  these  media 
when  they  are  sufficiently  separated  from  one  another  to 
permit  unhindered  development?  Note  the  color  and  inter- 
nal structure  of  the  colonies  as  well  as  their  relative  density. 
What  is  the  nature  of  the  surface  contour  and  arrangement 
of  the  colonies?  Note  their  general  character,  as  to  whether 
they  are  moist  or  dry,  compact  or  loosely  constructed, 
sharply  circumscribed  or  spreading  over  the  surface  of  the 
medium.  Do  the  gelatin  colonies  show  evidences  of  lique- 
faction? 

Agar-slant  Inoculations.— Observe  the  nature  of  the  growth 

on  the  surface  of  an  agar-agar-slant  inoculation.     Describe 

the  color,  texture,  and  optical  characters  of  the  growth. r  Is 

the  growth  confined  to  the  line  of  inoculation,  or  has  it  a 

13 


194  BACTERIOLOGY 

tendency  to  spread  over  the  surface  of  the  medium?  Is 
it  smooth  or  rough,  moist  or  dry,  glistening  or  dull  in 
character?  If  the  organism  forms  pigment,  note  whether 
the  pigment  is  confined  to  the  area  of  growth  or  whether  it 
extends  into  the  medium  itself.  Record  the  manner  in 
which  the  culture  changes  in  its  appearance  on  successive  days. 

Agar-stab  Inoculations. — Observe  the  nature  of  the  growth 
in  an  agar-agar-stab  inoculation.  Note  whether  the  growth 
is  most  voluminous  at  or  near  the  surface  or  in  the  depth 
of  the  stab.  If  the  organism  produces  pigment,  note  whether 
the  pigment  formation  is  most  marked  at  or  near  the  surface 
or  at  the  bottom  of  the  stab.  Record  the  alterations  that 
are  observed  on  several  successive  days. 

Gelatin-stab  Inoculations. — Observe  the  nature  of  the 
growth  in  a  gelatin-stab  inoculation.  Is  the  growth  most 
voluminous  at  or  near  the  surface  or  at  the  bottom  of  the 
stab?  Note  the  general  character  of  the  growth  on  the 
surface,  especially  as  to  its  contour,  extent,  and  color.  Note 
the  character  of  the  growth  in  the  stab.  Is  it  continuous 
along  the  whole  line  of  inoculation,  or  is  it  confined  to 
isolated  areas?  If  the  organism  has  the  property  of  liquefy- 
ing gelatin,  note  carefully  the  manner  in  which  the  lique- 
faction proceeds.  How  soon  does  liquefaction  begin,  and 
in  what  length  of  time  is  a  tube  of  gelatin  completely 
liquefied? 

Potato  Culture. — Observe  the  nature  of  the  growth  on 
potato.  This  is  an  important  differential  medium,  since 
some  organisms  grow  upon  it  very  sparingly  or  indeed 
almost  invisibly.  Other  organisms  grow  very  character- 
istically. Some  organisms  have  the  property  of  breaking 
up  the  starch  of  the  potato  into  simpler  compounds.  This 
is  sometimes  accompanied  by  the  evolution  of  gas.  Many 


BIOLOGIC  CHARACTERS  195 

of  the  chromogenic  bacteria  find  the  potato  a  most  suitable 
pabulum  on  which  to  form  their  pigment,  the  pigment 
formed  on  this  medium  having  at  times  an  especial  bril- 
liancy. Note  in  detail  all  the  changes  that  occur  in  the  growth 
on  successive  days. 

Growth  in  Bouillon. — Observe  whether  the  fluid  shows 
turbidity  or  not,  as  well  as  the  extent  and  distribution  of 
this  alteration.  Note  whether  any  sediment  is  being  formed, 
as  well  as  the  nature  and  amount  of  such  sediment.  Does 
the  organism  form  a  definite  growth  (pellicle  or  scum)  on 
the  surface  of  the  bouillon?  What  is  the  character  of  the 
pellicle?  Is  it  readily  dislodged,  and,  when  dislodged,  is 
it  replaced  by  a  new  pellicle?  Note  whether  the  color  of 
the  medium  has  become  altered.  Note  the  manner  in  which 
the  appearance  of  the  culture  changes  on  several  successive 
days. 

Growth  in  Litmus-milk. — Observe  the  nature  of  the  growth 
in  litmus-milk.  Has  the  reaction  of  the  medium  become 
altered?  To  what  is  such  alteration  attributable?  Note 
whether  there  is  precipitation  of  casein.  Record  the  extent 
and  rapidity  with  which  this  alteration  takes  placej  as  well 
as  the  reaction  of  the  fluid  while  the  change  is  being  pro- 
duced. Is  there  any  evidence  of  the  subsequent  liquefaction 
of  the  precipitated  casein?  Has  the  litmus  been  altered  in 
any  manner  except  as  shown  by  altered  reaction  of  the 
medium?  In  what  part  of  the  tube  has  such  alteration  of 
the  litmus  commenced?  If  the  litmus  has  been  decolorized, 
is  it  possible  to  restore  its  color  by  the  admixture  of  air  with 
the  fluid?  Note  the  order  in  which  the  appearance  of  the 
medium  ch'anges  on  successive  days. 

Growth  in  Special  Media. — The  special  culture  media  may 
be  employed  to  ascertain  additional  biologic  characters  of 


196  BACTERIOLOGY 

an  organism,  such  as  the  production  of  indol,  reduction  of 
nitrates  to  nitrites,  the  formation  of  ammonia,  production 
of  gas  in  media  containing  different  carbohydrates,  or  the 
reducing  power  of  the  organism  on  aniline  dyes,  etc. 

Influence  of  External  Agencies. — Note  the  vitality  of  the 
organism  under  the  influence  of  various  physical  and 
chemical  agents.  Determine  the  temperature  at  which  it 
thrives  best,  as  well  as  the  lowest  and  highest  temperatures 
at  which  growth  is  possible.  Determine  the  thermal  death- 
point  of  the  organism  by  subjecting  it  to  various  degrees 
of  temperature  from  55°  to  75°  C.  for  ten  minutes.  Deter- 
mine its  resistance  to  drying;  to  the  influence  of  light;  to 
the  influence  of  germicidal  substances.  Determine  the 
influence  of  different  gases  upon  the  growth  of  the  organisms, 
such  as  hydrogen,  nitrogen,  or  carbon  dioxide.  Determine 
the  chemical  reaction  of  the  culture  media  best  adapted  for 
its  growth.1 

BIOCHEMIC   CHARACTERS. 

If  the  organism  exhibits  chromogenic  properties,  ascertain 
whether  the  pigment  is  intra-  or  extracellular.  Ascertain 
under  what  conditions  of  temperature,  reaction,  and  con- 
stitution of  media,  or  under  what  atmospheric  conditions 
this  function  is  best  exhibited.  Note  the  influence  of  dif- 
ferent reagents  upon  the  pigment,  such  as  chloroform,  ether, 
alcohol,  water,  acids,  or  alkalies.  Note  whether  the  organism 
exhibits  photogenic  properties,  and  if  so,  ascertain  what 
conditions  are  most  suitable  for  the  manifestation  of  this 
phenomenon. 

1  For  more  detailed  description  of  the  variations  in  the  character  of 
the  macroscopic  and  microscopic  appearance  of  the  cultures  in  the  different 
media,  and  for  commonly  employed  terminology  the  student  is  referred  to 
Chester's  Determinative  Bacteriology  and  Eyre's  Bacteriologic  Technique, 


PATHOGENIC  PROPERTIES  197 

Ascertain  whether  the  organism  produces  enzymes.  Does 
it  manifest  a  proteolytic  function,  as  shown  by  the 
liquefaction  of  gelatin,  casein,  or  blood  serum?  Note 
whether  this  function  is  manifested  in  alkaline  or  in  acid 
condition  of  the  medium.  Does  it  manifest  a  precipitating 
effect  (rennet  ferment?)  upon  casein?  Note  whether  this 
is  manifested  in  alkaline  or  in  acid  condition  of  the 
medium.  Does  the  organism  have  the  property  of  breaking 
up  any  of  the  carbohydrates  into  simpler  compounds?  Is 
this  alteration  accompanied  or  not  by  the  liberation  of  gas? 
If  so,  ascertain  the  relative  amount  of  gas  formed  from  a 
given  quantity  of  carbohydrate.  Analyze  the  gas  formed, 
and  state  the  relative  proportion  of  carbon  dioxide  and 
residual  (explosive)  gas  formed. 

Ascertain  whether  the  organism  produces  indol.  Is  this 
substance  formed  with  the  simultaneous  reduction  of 
nitrates  to  nitrites?  Are  the  nitrites  reduced  further  into 
ammonia  ? 

PATHOGENIC  PROPERTIES. 

Ascertain  whether  any  of  the  animals  used  for  experi- 
mental purposes  are  susceptible  when  inoculated  with  the 
organism.  Are  all  species  of  laboratory  animals  equally 
susceptible,  or  are  some  immune?  Note  the  size  of  the 
dose  and  the  manner  of  inoculation  that  gives  the  most 
constant  and  characteristic  results.  What  are  the  symp- 
toms and  postmortem  appearances  produced?  What  is  the 
location  of  the  organisms  in  the  body  of  the  dead  animal  ? 
Are  they  confined  to  the  seat  of  inoculation,  or  are  they 
distributed  more  or  less  generally  throughout  the  body? 

Note  whether  the  virulence  of  the  organism  is  maintained 


198  BACTERIOLOGY 

when  grown  for  several  generations  on  artificial  media, 
or  whether  it  soon  becomes  attenuated.  Which  culture- 
medium  is  best  suited  to  conserve  the  virulence  of  the 
organism?  In  what  manner  does  its  environment  influence 
the  virulence?  If  the  virulence  is  readily  lost,  may  it  be 
regained  by  any  of  the  known  methods? 

Ascertain  whether  the  organism  forms  a  soluble  toxin 
when  grown  in  fluid  media,  as  sugar-free  bouillon.  If 
toxin  is  formed,  ascertain  whether  the  antitoxic  state  is 
readily  induced  in  susceptible  animals. 

If  no  soluble  toxin  is  formed,  ascertain  whether  animals 
may  be  immunized  by  the  injection  of  sub-lethal  doses 
of  dead  or  living  cultures.  Is  a  bactericidal  immunity 
induced  by  this  means?  Does  the  serum  of  immune  animals 
possess  protective  and  curative  properties  when  adminis- 
tered to  susceptible  animals  before  or  after  inoculation 
with  the  living  organism?  Does  the  serum  of  immune 
animals  possess  the  property  of  agglutinating  the  organ- 
isms in  relatively  higher  dilutions  than  the  serum  of  normal 
animals  of  the  same  species? 

The  majority  of  the  bacteria  may  be  identified  without 
resorting  to  such  a  detailed  study  of  the  biochemic  and 
pathogenic  properties  as  given  in  the  foregoing  outline,  but 
for  some  of  the  pathogenic  bacteria  it  has  been  necessary 
to  apply  all  the  known  tests  in  order  to  definitely  establish 
their  identity.  By  means  of  such  detailed  studies  on  related 
organisms,  it.  has  been  possible  to  differentiate  varieties 
whose  characters  are  constant,  yet  in  general  they  are  so 
closely  related  that  it  is  impossible  from  the  clinical  mani- 
festations produced  to  state  definitely  which  particular 
variety  of  organism  is  responsible  for  the  conditions. 


VARIATIONS  AND  VARIETIES  199 

VARIATIONS  AND  VARIETIES. 

As  in  the  case  of  all  other  living  things,  bacteria  are  modi- 
fied by  their  environment.  Such  modifications  manifest 
themselves  in  various  ways.  In  some  instances  they  are 
degenerative,  involving  alterations  in  form  and  function 
that  are  easily  detected  by  appropriate  methods  of  exami- 
nation. Often  such  changes  are  but  transitory  and  are 
referable  to  the  influences  of  well-known  causes,  the  removal 
of  which  permits  the  bacteria  to  resume  their  normal  state. 
(See  Involution  Forms.)  In  other  instances  more  or  less 
prolonged  environmental  influences,  of  which  we  know  but 
little,  appear  to  have  brought  about  alterations  in  function 
with  no  appreciable  changes  in  form.  Sometimes  the  one 
or  the  other  of  such  modifications  may  be  brought  about 
at  will  by  appropriate  experimental  methods. 

From  the  early  days  of  modern  bacteriology  confusion 
has  arisen  at  times  in  connection  with  the  establishment  of 
definite  species. 

It  was  frequently  found  that  among  the  species,  as  deter- 
mined by  methods  then  available,  individual  members  of 
a  species  would  exhibit  variations  in  particular  functions 
that  differentiated  them  from  the  accepted  type.  Some- 
times these  differences  were  morphological,  more  often  they 
were  physiological.  Occasionally  they  could  be  detected  by 
crude  culture  methods  then  in  vogue — more  often — as  the 
studies  progressed,  they  were  demonstrable  only  by  more 
refined  special  methods.  To  recall  this  confusion  one  need 
but  mention  the  marked  functional  variations  of  Bacillus 
coli  communis  and  the  striking  morphological  differences 
seen  in  Bacillus  diphtherise. 

By  the  discovery  of  methods  better  suited  to  bring  out 


200  BACTERIOLOGY 

finer  differences — notably,  those  that  took  into  considera- 
tion the  zymogenic  powers  of  bacteria — it  was  soon  possible 
to  speak  of  groups,  strains,  or  types  among  the  species,  one 
strain  or  group  differing  from  another  in  its  ability  to  ferment 
certain  carbohydrates,  with  recognizable  end-products,  while 
other  strains  were  devoid  of  this  power,  though  in  all  other 
particulars  the  two  strains  may  have  been  identical.  By 
the  application  of  such  tests  many  species  have  been  sepa- 
rated into  groups  and  some  groups  into  sub-groups;  some 
fermenting  all  sugars,  others  fermenting  only  particular 
sugars.  Some  fermenting  with  free  gases  as  an  end-product 
others  with  no  gas  but  only  acids  as  end-products.  These 
functions  are  subject  to  quantitative  variations  and  in  a 
few  cases  they  may  temporarily  disappear,  and  occasionally 
by  experimental  methods,  may  be  made  to  disappear,  but 
as  yet  it  has  not  been  possible  by  any  known  method  to 
endow  a  species,  by  nature  devoid  of  the  power  to  ferment 
sugar,  with  such  power. 

The  ability  of  a  pathogenic  species  to  cause  in  animals 
pathological  lesion  identical  to  those  from  which  the  species 
was  obtained  was  held  for  a  long  time  as  the  test  par  excel- 
lence for  the  identification  of  pathogenic  species.  Accord- 
ing to  the  standards  then  in  common  use  two  cultures  from 
different  cases  of  the  same  disease  may  have  been  identical 
in  all  other  particulars,  yet  if  one  was  capable  of  reproducing 
in  animals  lesions  similar  to  those  in  man  from  which  that 
culture  was  obtained  and  the  other  was  devoid  of  that 
power,  they  were  generally  regarded  as  two  distinct  species. 

To  illustrate  we  have  only  to  recall  the  confusion  that 
existed  in  connection  with  the  diphtheria  bacillus  and  the 
several  "pseudo"  diphtheria  bacilli  that  were  described. 
We  now  know  that  variations  in  pathogenic  power  is 


VARIATIONS  AND  VARIETIES  201 

one  of  the  commonest  phenomena  noticed  among  disease- 
producing  bacteria.  And  we  also  know  that  by  artificial 
procedures  many  of  the  highly;  pathogenic  organisms  may 
be  in  part  or  wholly  deprived  of  their  powers  to  cause  the 
lesions  peculiar  to  the  activities  of  the  normal  organisms, 
retaining  at  the  same  time  all  other  peculiarities  common  to 
the  species. 

The  manifold  studies  upon  infection  and  immunity  have 
placed  at  our  disposal  methods  by  which  it  is  possible  to 
detect  differences  among  closely  related  types  of  the  same 
species  that  cannot  be  revealed  in  any  other  way. 

Such  differences  appear  to  be  idioplasmic,  if  the  word  is 
appropriate  to  bacteria,  and  though  slight  quantitative 
fluctuations  may  be  noted,  the  strains  characterized  by  them 
have  them  as  fixed,  inherent  peculiarities. 

We  may  regard  them  therefore  not  as  indicating  modi- 
fications of  a  component  common  to  the  species,  but  rather 
as  specific  components  possessed  by  some  varieties  of  a 
species  and  not  by  others;  not  necessarily  as  new  characters 
evolving  from  environmental  influences,  insofar  as  can  be 
determined,  but  as  natural,  fundamental  components  revealed 
only  by  newer  adequate  methods  of  investigation. 

Through  the  use  of  certain  immunologic  methods  or  tests 
it  is  now  possible  to  subdivide  most  of  the  pathogenic  species 
into  distinct  sub-groups — the  one  group  differing  from  the 
other  only  in  the  presence  or  absence  of  those  components 
necessary  to  complete  the  differential  reactions. 

To  make  this  clear:  If  one  immunize  an  animal  from 
Bacillus  typhosus  the  blood  serum  of  such  animal  if  brought 
together  with  Bacillus  typhosus  causes  the  bacilli  to  gather 
in  distinct  clumps,  whereas,  if  such  serum  be  brought  in 
contact  with  Bacillus  coli  communis,  or  with  Bacillus  dysen- 


202  BACTERIOLOGY 

terise,  both  closely  related  to  Bacillus  typhosus,  no  such 
clumping  will  occur. 

Again,  if  we  mix  with  that  serum  typhoid  bacilli  derived 
from  any  number  of  cases  of  typhoid  fever  the  serum  will 
cause  clumping  of  some  cultures  and  not  others,  even  though 
all  cultures  came  from  individuals  having  the  same  disease 
and  by  the  common  tests  all  are  alike. 

We  note  here  a  high  degree  of  specificity — not  only  a 
specificity  peculiar  to  certain  species  but  likewise  peculiar 
to  certain  individual  members  of  the  same  species.  We  are 
justified  then  in  concluding  that,  from  the  standpoint  of 
this  test,  all  the  cultures  of  typhoid  bacilli  that  were  clumped 
by  the  serum  used  were  of  the  same  strain  or  type  as  that 
used  in  immunizing  the  animal;  while  all  those  that  did  not 
clump  with  the  same  serum  were  of  different  strains  or 
types.  If  we  now  immunize  an  animal  from  anyone  of 
this  latter  group  of  typhoid  bacilli  we  shall  find  that  the 
serum  will  cause  clumping  of  the  bacilli  in  the  culture  used 
for  immunization  and  will  probably  react  in  the  same 
manner  with  some  of  the  other  cultures — but  not  with  all, 
nor  with  the  cultures  embraced  by  our  first  group.  Thus, 
we  will  have  established  at  least  two  groups,  or  types  that 
have  distinct,  specific  serologic  reactions,  and  so  we  may 
go  on  and  perhaps  establish  additional  groups. 

This  reaction  commonly  known  as  the  "agglutination 
reaction"  is  invaluable  in  the  efforts  to  assemble  species  into 
groups  and  subgroups  or  types  specifically  different — the 
one  from  the  other  insofar  as  the  reaction  goes. 

If  by  this  procedure  we  find  that  two  groups — A  and  B  of  an 
infective  species — can  be  established  and  with  the  members  of 
Type  A  we  render  an  animal  so  highly  immune  that  its  serum 
may  be  expected  to  possess  curative  properties  for  the  disease 


VARIATIONS  AND  VARIETIES    .  203 

caused  by  the  bacterial  species  under  consideration,  we  may 
find  it  to  be  curative  or  preventive  for  all  Type  A  infections  and 
not  at  all  so  or  only  lowly  so  for  Type  B  infections,  and 
vice  versa.  In  other  words  those  specific  components  of  the 
members  of  Type  A  which  are  revealed  by  the  agglutination 
test  may  indicate  the  possession  by  Type  A  organisms  of  com- 
ponents that  call  forth  the  elaboration  by  the  tissues  of  the 
immunized  animal  of  bodies  that  neutralize  only  the  poisons 
of  the  bacteria  of  Type  A — that  is,  of  the  particular  type 
from  which  the  animal  was  rendered  immune. 

This  is  a  point  of  fundamental  value  in  connection  with 
the  use  of  antisera  for  the  cure  of  infections.  For  the  best 
results  such  antisera  must  be  homologously  related  to  the 
"type"  organisms  concerned  in  the  infection  for  which  it 
is  to  be  employed. 

As  the  result  of  all  this,  bacteriologists  are  today  concern- 
ing themselves  more  with  groups  and  group  reactions  than 
with  individual  species  and  their  peculiarities;  that  is  to  say 
the  "typing"  of  bacteria  has  become  one  of  the  routine 
operations  in  bacteriological  laboratories,  in  consequence  we 
speak  of  the  "colon  type,"  the  "dysenteric  type,"  the 
"  pneumococcus  type,"  etc.,  meaning  that  the  particular 
organism  or  culture  with  which  we  are  dealing  is  a  species 
belonging  to  one  or  the  other  "type"  as  determined  by  its 
agglutinability,  and  it  may  or  may  not  conform  to  all  the 
other  reactions  by  which  the  so-called  typical  species  is 
identified.  • 

Working  from  such  a  standpoint  we  now  know  that 
many  of  the  important  disease-producing  organisms  lend 
themselves  to  such  grouping,  and  by  the  adoption  of  this 
plan  of  work  it  has  been  possible  to  make  advances  and  to 
interpret  phenomena  otherwise  impossible. 


204  BACTERIOLOGY 

By  using  the  agglutination  test  we  now  know  that  the 
organisms  causing  pneumonia  may  be  definitely  subdivided 
into  at  least  four  groups;  that  the  pathogenic  streptococci, 
fall  into  at  least  two,  possibly  four  groups;  that  several 
strains  of  dysenteric  bacilli  and  at  least  two  strains  of 
meningococci  have  been  demonstrated.  And  throughout  we 
find  a  constant  specific  relationship  between  strains  and 
their  homologous  antibodies.  To  repeat:  This  is  a  matter 
of  the  greatest  practical  moment,  for  the  serum  from  an 
animal  that  is  immune  from  the  members  of  one  group,  while 
it  may  possess  high  potency  if  used  against  infections  caused 
by  members  of  that  group,  may  be  of  but  little  or  no  value 
if  used  in  the  treatment  of  infections  caused  by  members  of 
another  closely  related  group.  (See  "  Pneumococcus "  and 
"Streptococcus"  paragraphs  on  variations.) 

MICROSCOPIC   EXAMINATION   OF   PREPARATIONS. 

The  Different  Parts  of  the  Microscope. — Before  describing 
the  method  of  examining  preparations  microscopically,  a 
few  definitions  of  the  terms  used  in  connection  with  the 
microscope  may  not  be  out  of  place.  (The  different  parts 
of  the  microscope  referred  to  below  are  indicated  by  letters 
in  Fig.  34.) 

The  ocular  or  eye-piece  (A)  is  the  lens  at  which  the  eye  is 
placed  when  looking  through  the  instrument.  It  serves  to 
magnify  the  image  projected  through  the  objective. 

The  objective  (B)  is  the  lens  which  is  at  the  distal  end  of 
the  barrel  of  the  instrument,  and  which  serves  to  magnify 
the  object  to  be  examined. 

The  stage  (c)  is  the  shelf  or  platform  of  the  microscope  on 
which  the  object  to  be  examined  rests. 


MICROSCOPIC  EXAMINATION  OF  PREPARATIONS     205 

The  diaphragms  are  the  perforated  stops  that  fit  in  the 
center  of  the  stage.     They  vary  in  size,  so  that  different 


FIG.  34 


— G 


amounts  of  light  may  be  admitted  to  the  object  by  using 
diaphragms  with  larger  or  smaller  openings. 


206  BACTERIOLOGY 

The  "iris"  diaphragm  (D)  opens  and  closes  like  the  iris 
of  the  eye.  It  is  so  arranged  that  its  opening  for  admission 
of  light  can  be  increased  or  diminished  by  moving  a  small 
lever  in  one  or  another  direction. 

The  reflector  (E)  is  the  mirror  placed  beneath  the  stage, 
which  serves  to  illuminate  the  object  to  be  examined. 

The  coarse  adjustment  (F)  is  the  rack-and-pinion  arrange- 
ment by  which  the  barrel  of  the  microscope  can  be  quickly 
raised  or  lowered. 

The  fine  adjustment  (G)  serves  to  raise  and  lower  the 
barrel  of  the  instrument  very  slowly  and  gradually. 

For  the  microscopic  study  of  bacteria  it  is  essential  that 
the  microscope  be  provided  with  an  oil-immersion  system 
and  a  sub-stage  condensing  apparatus. 

The  oil-immersion  or  homogeneous  system  consists  of  an 
objective  so  constructed  that  it  can  only  be  used  when  the 
transparent  media  through  which  the  light  passes  in  enter- 
ing it  are  all  of  the  same  index  of  refraction — i.  e.,  are 
homogeneous.  This  is  accomplished  by  interposing  between 
the  face  of  the  lens  and  the  cover-slip  covering  the  object 
to  be  examined  a  body  which  refracts  the  light  in  the  same 
way  as  do  the  glass  slide,  the  cover-slip,  and  the  glass  of 
which  the  objective  is  made.  For  this  purpose,  a  drop  of 
oil  of  the  same  index  of  refraction  as  the  glass  is  placed  upon 
the  face  of  the  lens,  and  the  examinations  are  made  through 
this  oil.  There  is  thus  little  or  no  loss  of  light  from  deflec- 
tion, as  is  the  case  in  the  dry  system. 

The  sub-stage  condensing  apparatus  (H)  is  a  system  of 
lenses  situated  beneath  the  central  opening  of  the  stage. 
They  serve  to  condense  the  light  passing  from  the  reflector 
to  the  object  in  such  a  way  that  it  is  focussed  upon  the 
object,  thus  furnishing  the  greatest  amount  of  illumination. 


MICROSCOPIC  EXAMINATION  OF  PREPARATIONS     207 

Between  the  condenser  and  reflector  is  placed  the  "iris" 
diaphragm,  the  aperature  of  which  can  be  regulated,  as 
circumstances  require,  to  permit  of  either  a  very  small  or 
a  very  large  amount  of  light  passing  to  the  object. 

The  nose-piece  (i)  consists  of  a  collar,  or  group  of  collars 
joined  together  (two  or  more),  that  is  attached  to  the  distal 
end  of  the  tube  of  the  microscope.  It  enables  one  to  attach 
several  objectives  to  the  instrument  in  such  a  way  that  by 
simply  rotating  the  nose-piece  the  various  lenses  of  different 
power  may  be  conveniently  used  in  succession. 

Dark-field  Illumination. — This  refers  to  a  result  obtained 
through  the  use  of  an  apparatus  that  so  deflects  and  reflects 
the  light's  rays  that  the  field  is  dark  and  the  objects  in  it 
brilliantly  light.  It  is  used  only  for  the  examination  of 
unstained  living  objects  and  is  capable  of  revealing  the 
most  minute  particles  and  microorganisms.  It  is  especially 
useful  for  the  study  of  the  normal  morphology  and  move- 
ment of  spirochete  and  trypanosomes  and  for  the  detection 
of  bodies  so  small  or  otherwise  so  constituted  as  not  to  be 
visible  by  the  ordinary  methods  of  microscopic  examination. 
Two  forms  of  the  illuminator  are  in  use — one  that  slips  into 
the  collar  ordinarily  carrying  the  sub-stage  condensing 
apparatus,  the  other  is  made  in  the  form  of  a  slide  and  is 
placed  on  the  stage  directly  over  the  opening  for  illumina- 
tion. Both  provide  for  the  complete  cutting  off  of  direct 
central  rays  of  light,  allowing  only  the  lateral  rays  to  reach 
the  objects  and  be  reflected  by  them  to  the  eye.  Both  require 
very  intense  illumination  for  the  best  results.  This  may  be 
obtained  from  a  Welsbach  burner,  or  a  small  arc  or  incan- 
descent light.  In  both  cases  the  light's  rays  must  be 
condensed  upon  the  reflector  of  the  microscope  by  means 
of  a  condensing  lens. 


208  BACTERIOLOGY 

Microscopic  Examination  of  Cover-slips. — The  stained  cover- 
slip  is  to  be  examined  with  the  oil-immersion  objective, 
and  with  the  diaphragm  of  the  sub-stage  condensing  appara- 
tus open  to  its  full  extent.  The  object  gained  by  allowing 
the  light  to  enter  in  such  a  large  volume  is  that  the  contrast 
produced  by  the  colored  bacteria  in  the  brightly  illuminated 
field  is  much  more  conspicuous  than  when  a  smaller  amount 
of  light  is  thrown  upon  them.  This  is  true  not  only  for 
stained  bacteria  on  cover-slips,  but  likewise  for  their  differen- 
tiation from  surrounding  objects  when  they  are  located  in 
tissues.  With  unstained  bacteria  and  tissues,  on  the  con- 
trary, the  structure  can  best  be  made  out  by  reducing  the 
bundle  of  light-rays  to  the  smallest  amount  compatible  with 
distinct  vision,  and  in  this  way  favoring,  not  color-contrast, 
but  contrasts  which  appear  as  lights  and  shadows,  due  to  the 
differences  in  permeability  to  light  of  the  various  parts  of 
the  material  under  examination. 

Steps  in  Examining  Stained  Preparations  with  the  Oil-immer- 
sion System. — Place  upon  the  center  of  the  cover-slip  which 
covers  the  preparation  a  small  drop  of  immersion  oil.  Place 
the  slide  upon  the  center  of  the  stage  of  the  microscope. 
With  the  coarse  adjustment  lower  the  oil-immersion  objec- 
tive until  it  just  touches  the  drop  of  oil.  Open  the  illumi- 
nating apparatus  to  its  full  extent.  Then,  with  the  eye 
to  the  ocular  and  the  hand  on  the  fine  adjustment,  turn  the 
adjusting  screw  toward  the  right  until  the  field  becomes 
somewhat  colored  in  appearance.  When  this  is  seen  pro- 
ceed more  slowly  in  the  same  direction,  and,  after  one  or 
two  turns,  the  object  will  be  in  focus.  Do  not  remove  the 
eye  from  the  instrument  until  this  has  been  accomplished. 

Then,  with  one  hand  upon  the  fine  adjustment  and  the 
thumb  and  index  finger  of  the  other  hand  holding  the  slide 


MICROSCOPIC  EXAMINATION  OF  PREPARATIONS    209 

lightly  by  its  end,  it  may  be  moved  about  under  the  objec- 
tive. At  the  same  time  the  screw  of  the  fine  adjustment 
must  be  turned  back  and  forth,  so  that  the  different  planes 
of  the  preparation  may  be  brought  into  focus  one  after  the 
other.  In  this  way  the  whole  section  or  preparation  may 
be  inspected.  When  the  examination  is  finished  raise  the 
objective  from  the  preparation  by  turning  the  screw  of  the 
coarse  adjustment  toward  you.  Remove  the  preparation 
from  the  stage,  and,  with  a  fine  silk  cloth  or  handkerchief, 
wipe  very  gently  and  carefully  the  oil  from  the  face  of  the  lens. 

During  work,  of  course,  the  lens  need  not  be  cleaned  and 
put  away  after  each  examination;  but  when  the  work  for 
the  day  is  over  an  immersion  lens  is  best  protected  in  this 
way.  Under  no  circumstances  should  it  be  allowed  to  remain 
in  the  immersion  oil  or  vexposed  to  dust  for  any  length  of 
time. 

Examination  of  Unstained  Preparations. — "Hanging  drops." 
It  frequently  becomes  necessary  to  examine  bacteria  in  the 
unstained  condition.  The  circumstances  calling  for  this 
arise  while  studying  the  multiplication  of  cells,  the  germina- 
tion of  spores,  and  the  absence  or  presence  of  motility. 

In  this  method  the  organisms  to  be  studied  are  suspended 
in  a  drop  of  physiological  salt  solution  or  of  bouillon,  or  a 
tiny  drop  of  either  agar-agar  or  gelatin,  inoculated  with 
the  organism,  may  be  employed.  The  drop  is  placed  in  the 
center  of  a  clean  cover-slip  which  has  been  sterilized  in  the 
flame  and  which  is  then  inverted  over  the  depression  in 
a  sterilized  so-called  "hollow-ground"  slide  to  which  it  is 
sealed  with  vaseline.  A  convenient  and  quick  method  of 
making  the  preparation  is,  after  placing  the  drop  in  the 
center  of  the  cover-slip,  to  invert  over  it  the  slide,  around 
the  depression  in  which  a  ring  of  vaseline  has  been  painted. 
14 


210  BACTERIOLOGY 

The  slip  adheres  and  the  preparation  may  then  be  handled 
without  fear  of  disturbing  the  drop  or  the  position  of  the 
slip  over  the  depression.  When  completed  it  has  the  appear- 
ance shown  in  Fig.  35.  The  drop  hangs  in  an  air-tight 
chamber  so  that  both  evaporation  and  contamination  are 
prevented. 

This  is  known  as  the  "hanging-drop"  method  of  exami- 
nation or  cultivation.  It  is  indispensable  for  the  purposes 
mentioned,  and  at  the  same  time  requires  considerable  care 
in  its  manipulation.  The  fluid  is  so  transparent  that  the 
cover-slip  may  be  broken  by  the  objective  being  brought 
down  upon  the  preparation  before  one  is  aware  that  the 
focal  distance  has  been  reached.  This  may  be  avoided  by 

FIG.  35 


Longitudinal  section  of  hollow-ground  glass  slide  for  observing  bacteria  in 
hanging  drops. 

bringing  the  edge  of  the  drop  into  the  center  of  the  field  with 
one  of  the  higher  power  dry  lenses.  When  this  is  accomplished 
substitute  the  immersion  for  the  dry  system,  when  the  edge 
of  the  drop  is  readily  detected  with  the  higher  power  lens 
somewhere  near  the  centre  of  the  field. 

In  examining  bacteria  by  this  method  there  is  a  possibility 
of  error  that  must  be  guarded  against.  All  microscopic 
insoluble  particles  in  suspension  in  fluids  possess  a  peculiar 
tremor  or  vibratory  motion,  the  so-called  "Brownian 
motion."  This  is  very  apt  to  give  the  impression  that  the 
organisms  under  examination  are  motile,  when  in  truth  they 
are  not  so,  their  movement  in  the  fluid  being  only  this 
molecular  tremor. 


MICROSCOPIC  EXAMINATION  OF  PREPARATIONS      211 

The  difference  between  the  motion  of  bodies  undergoing 
this  molecular  tremor  and  that  possessed  by  certain  living 
bacteria  is  that  the  former  particles  never  move  from  their 
place  in  the  field,  while  living  motile  bacteria  alter  their 
position  in  relation  to  the  surrounding  organisms,  and  may 
dart  from  one  position  in  the  field  to  another.  In  some  cases 
the  true  movement  of  bacteria  is  very  slow  and  undulating, 
while  in  others  it  is  rapid  and  darting.  The  molecular 
tremor  may  be  seen  with  non-motile  and  with  dead  organisms. 

NOTE. — Prepare  three  hanging-drop  preparations — one 
from  a  drop  of  dilute  India-ink,  a  second  from  a  culture  of 
micrococci,  and  a  third  from  a  culture  of  the  bacillus  of 
typhoid  fever.  In  what  way  do  they  differ? 

Study  of  Spore  Formation. — The  hanging-drop  method  just 
mentioned  is  not  only  employed  for  detecting  the  motility 
of  an  organism,  but  also  for  the  study  of  its  mode  of  spore- 
formation. 

Since  with  aerobic  organisms  spore  formation  occurs,  as 
a  rule,  only  in  the  presence  of  oxygen,  and  is  induced  more 
by  limitation  of  the  nutrition  of  the  organisms  than  by  any 
other  factor,  it  is  essential  that  these  two  points  should  be 
borne  in  mind  in  preparing  the  drop  cultures  in  which  the 
process  is  to  be  studied.  For  this  reason  the  drop  of  bouillon 
should  be  small  and  the  air  chamber  relatively  large. 

A  very  thin  drop  of  sterilized  agar-agar  may  be  substi- 
tuted for  the  bouillon. »  It  serves  to  retain  the  organisms  in 
a  fixed  position,  and  the  process  may  be  more  easily  followed. 
.  As  soon  as  finished  the  preparation  is  to  be  examined 
microscopically  and  the  condition  of  the  organisms  noted. 
It  is  then  to  be  retained  in  a  warm  chamber,  and  kept  under 


212  BACTERIOLOGY 

continuous  observation.  The  form  of  chamber  best  adapted 
to  the  purpose  is  one  which  envelops  the  whole  microscope. 
It  is  provided  with  a  window  through  which  the  light  enters, 
and  an  arrangement  by  which  the  slide  may  be  moved  from 
the  outside.  The  formation  of  spores  requires  a  much 
longer  time  than  the  germination  of  spores  into  bacilli,  but 
with  patience  both  processes  may  be  satisfactorily  observed. 

It  will  be  noticed  that  the  description  of  this  process  is 
very  much  like  that  which  immediately  precedes,  but  differs 
from  it  in  one  respect,  viz.,  that  in  this  manipulation  we 
are  not  making  a  preparation  which  is  simply  to  be  ex- 
amined and  then  thrown  aside,  but  it  is  an  actual  pure 
culture,  and  must  be  kept  as  such,  otherwise  the  observa- 
tion will  be  worthless.  For  this  reason  the  greatest  care 
must  be  observed  in  the  sterilization  of  all  objects  employed. 
Studies  upon  spore  formation  by  this  method  frequently 
continue  over  hours,  and  sometimes  days,  and  contamina- 
tion must,  therefore,  be  carefully  guarded  against.  The 
study  should  be  begun  with  the  vegetative  form  of  the 
organisms;  the  hanging-drop  preparation  should,  for  this 
reason,  always  be  made  from  a  perfectly  fresh  culture  of 
the  organism  under  consideration  before  time  has  elapsed 
for  spores  to  form. 

The  simple  detection  of  the  presence  or  absence  of  spore 
formation  can  in  many  cases  be  made  by  other  methods. 
For  example,  many  species  of  bacteria  which  possess  this 
property  form  spores  most  readily  upon  media  from  which 
it  is  somewhat  difficult  for  them  to  obtain  the  necessary 
nourishment;  potatoes  and  agar-agar  that  have  become 
a  little  dry  offer  very  favorable  conditions,  because  of  the 
limited  area  from  which  the  growing  bacteria  can  draw  their 
nutritive  supplies,  and  because  of  the  free  access  which  they 


MICROSCOPIC  EXAMINATION  OF  PREPARATIONS     213 

have  to  oxygen,  for,  their  growth  being  on  the  surface,  they 
are  surrounded  by  this  gas  unless  means  are  taken  to  prevent 
it.  By  the  hanging-drop  method,  however,  more  than  -this 
specific  property  may  be  determined.  It  is  possible  not  only 
to  detect  the  stages  and  steps  in  the  formation  of  endogenous 
spores,  but  when  the  spores  are  completely  formed  their 
germination  into  mature  rods  may  be  seen  by  transferring 
them  to  a  fresh  bouillon-drop  or  drop  of  agar-agar  preserved 
in  the  same  way.  The  word  rods  is  used  because  we  have  as 
yet  no  evidence  that,  endogenous  spore-formation  occurs 
in  any  of  the  other  morphological  groups  or  bacteria. 

Hanging-block  Cultures. — Hill1  has  devised  a  method  for 
observing  the  development  of  individual  bacteria,  which 
consists  in  the  substitution  for  the  ordinary  "hanging  drop" 
of  liquid  or  jelly  a  cube  of  solidified  agar-agar,  on  the  surface 
of  which  the  bacteria  are  distributed. 

The  "hanging  block"  i's  prepared  as  follows:  "Pour 
melted  nutrient  agar  into  a  Petri  dish  to  the  depth  of  one- 
eighth  to  one-quarter  inch.  Cool  this  agar  and  cut  from  it 
a  block  about  one-quarter  to  one-third  inch  square  and  of 
the  thickness  of  the  layer  of  agar  in  the  dish.  This  block  has 
a  smooth  upper  and  under  surface.  Place  it,  under  surface 
down,  on  a  slide  and  protect  it  from  dust.  Prepare  an 
emulsion  in  sterile  water  of  the  organism  to  be  examined  if 
it  has  been  grown  on  a  solid  medium,  or  use  a  broth  culture; 
spread  the  emulsion  or  broth  upon  the  upper  surface  of  the 
block,  as  if  making  an  ordinary  cover-slip  preparation. 
Keep  the  slide  and  block  in  an  incubator  at  37°  C.  for  five 
to  ten  minutes  to  dry  slightly.  Then  lay  a  clean  sterile 
cover-slip  on  the  inoculated  surface  of  the  block  in  close 
contact  with  it,  avoiding,  if  possible,  the  formation  of  air- 

1  Journal  of  Medical  Research,  1902,  vol.  vii,  p.  202. 


214  BACTERIOLOGY 

bubbles.  Remove  the  slide  from  the  lower  surface  of  the 
block,  and  invert  the  cover-slip  so  that  the  agar-block  is 
uppermost.  With  a  platinum  loop  run  a  drop  or  two  of 
melted  agar  along  each  side  of  the  agar  block  where  it  is 
in  contact  with  the  cover-slip.  This  seal  hardens  at  once, 
preventing  slipping  of  the  block.  Place  the  preparation  in 
the  incubator  again  for  five  or  ten  minutes  to  dry  the  agar 
seal.  Invert  this  preparation  over  a  moist  chamber  and  seal 
the  cover-slip  in  place  with  white  wax  or  paraffin.  Vaseline 
softens  too  readily  at  37°  C.,  allowing  shifting  of  the  cover- 
slip.  The  preparation  may  then  be  examined  at  leisure." 

Aerobic  bacteria  receive  sufficient  oxygen  by  diffusion, 
and  for  anaerobic  bacteria  it  will  suffice  to  hang  the  block 
in  a  chamber  containing  a  little  alkaline  pyrogallic  acid  solu- 
tion. This  absorbs  all  oxygen. 

Study  of  <Jelatin  Cultures. — As  has  been  previously  stated, 
the  behavior  of  bacteria  toward  gelatin  differs — some  of 
them  producing  apparently  no  alteration  in  the  medium, 
while  the  growth  of  others  is  accompanied  by  an  enzymotic 
action  that  results  in  liquefaction  of  the  gelatin  at  and 
around  the  place  at  which  the  colonies  are  growing.  In 
some  instances  this  liquefaction  spreads  laterally  and  down- 
ward, causing  a  saucer-shaped  excavation;  while  in  others 
the  colony  sinks  almost  vertically  into  the  gelatin  and  may 
be  seen  lying  at  the  bottom  of  a  funnel-shaped  depression. 
These  differences  are  constantly  employed  as  one  of  the 
means  of  differentiating  otherwise  closely  allied  species  and 
varieties.  (See  Fig.  32.)  Studies  upon  the  spirillum  of 
Asiatic  cholera  and  a  number  of  kindred  species,  for 
example,  reveal  decided  differences  in  the  form  of  lique- 
faction produced  by  these  various  organisms.  The  minutest 
detail  in  this  respect  must  be  noted,  and  its  frequency  or 
constancy  under  varying  conditions  determined. 


CHANGES  IN  THE  REACTION  OF  MEDIA          215 

Cultures  on  Potato. — A  useful  factor  in  the  identification 
of  an  organism  is  its  growth  on  sterilized  potato.  Many 
organisms  present  appearances  under  this  method  of  cul- 
tivation which  alone  can  almost  be  considered  characteristic. 
In  some  cases  coarsely  lobulated,  elevated,  dry  or  moist 
patches  of  development  occur  after  a  few  hours;  again,  the 
growth  may  be  finely  granular  and  but  slightly  elevated 
above  the  surface  of  the  potato;  at  one  time  it  will  be  dry 
and  dull  in  appearance,  again  it  may  be  moist  and  glisten- 
ing. Sometimes  bubbles,  due  to  the  fermentative  action  of 
the  growing  bacteria  on  the  carbohydrates  of  the  potato, 
are  produced. 

A  most  striking  form  of  development  on  potato  is  that 
often  exhibited  by  the  bacillus  of  typhoid  fever  and  the 
bacillus  of  diphtheria.  After  inoculation  of  a  potato  with 
either  of  these  organisms  there  is  usually  no  naked-eye 
evidence  of  growth,  though  microscopic  examination  of 
scrapings  from  the  surface  of  the  potato  reveals  an  active 
multiplication  of  the  organisms  which  had  been  planted 
there.  The  potato  is  one  of  the  first  of  the  differential  media. 

CHANGES  IN  THE  REACTION  OF  MEDIA  AS  A  RESULT 
OF  BACTERIAL  ACTIVITY. 

For  purposes  of  differentiation,  much  stress  is  laid  upon 
the  reaction  assumed  by  media  as  a  result  of  bacterial 
growth.  Under  the  influence  of  certain  species  the  medium 
will  become  acid,  under  that  of  others  it  is  alkaline,  while 
some  cause  little  or  no  change.  In  media  of  particular 
composition — i.  e.,  those  containing  traces  of  fermentable 
carbohydrates,  notably  muscle-sugar,  as  seen  in  infusions 
of  fresh  meat — the  reaction  may  become  acid  with  the  begin- 


216  BACTERIOLOGY 

ning  of  growth  and  subsequently  change  to  alkaline  after 
the  supply  of  fermentable  sugar  is  exhausted.  These  changes 
of  reaction  are  most  conveniently  observed  through  the  use 
of  indicators — bodies  that  either  lose  or  change  their  usual 
color  as  the  reaction  of  the  medium  to  which  they  are  added 
changes. 

Such  substances  as  litmus,  in  the  form  of  the  so-called 
"litmus  tincture,"  and  coralline  (rosolic  acid)  in  alcoholic 
solution,  have  been  commonly  employed  for  this  purpose, 
though  many  other  indicators  having  definite  ranges  of 
usefulness  are  now  being  employed.  (See  paragraphs  on 
Reaction.)  They  may  be  added  to  the  media  in  the  pro- 
portions given  in  the  chapter  on  Media,  and  the  changes  in 
their  colors  studied  with  different  bacteria.  Milk  and  litmus 
tincture  or  peptone  solution  to  which  rosolic  acid  has  been 
added  are  excellent  media  for  this  experiment. 

Fermentation. — The  production  of  gas  as  an  indication  of 
fermentation  is  an  accompaniment  of  the  growth  of  certain 
bacteria.  This  is  best  studied  in  media  to  which  1  to  2  per 
cent,  of  grape-sugar  (glucose)  has  been  added.  A  convenient 
method  of  demonstrating  this  property  is  to  employ  a  tube 
about  half  full  of  agar-agar  containing  the  necessary  amount 
of  grape-sugar.  The  medium  is  to  be  liquefied  on  a  water- 
bath,  and  then  cooled  to  about  42°  C.,  when  a  small  quantity 
of  a  pure  culture  of  the  organism  under  consideration  should 
carefully  be  distributed  through  it.  The  tube  is  then  placed 
in  ice-water  and  rapidly  solidified  in  the  vertical  position. 
When  solid  it  is  placed  in  the  incubator.  After  twenty-four 
to  thirty-six  hours,  if  the  organism  possesses  the  property 
of  causing  fermentation  of  glucose,  the  medium  will  be 
dotted  everywhere  with  very  small  cavities  containing  the 
gas  that  has  resulted. 


CHANGES  IN  THE  REACTION  OF  MEDIA          217 

This  property  of  fermentation  with  evolution  of  gas  is  of 
such  importance  as  a  differential  characteristic  that  con- 
siderable attention  has  been  given  to  it,  and  those  who  have 
been  most  intimately  concerned  in  the  development  of  our 
knowledge  on  the  subject  do  not  consider  it  sufficient  to 
say  that  the  growth  of  an  organism  "is  accompanied  by  the 
production  of  gas-bubbles/'  but  that  under  given  condi- 
tions we  should  determine  not  only  the  amount  of  gas  or 
gases  produced  by  the  organism  under  consideration,  but 
also  their  nature.  For  this  purpose,  Smith1  recommends  the 
employment  of  the  fermentation  tube.  This  is  a  tube  bent 
at  an  acute  angle,  closed  at  one  end  and  enlarged  with  a  bulb 
at  the  other.  At  the  bend  the  tube  is  constricted.  To  it 
a  glass  foot  is  attached  so  that  the  tube  may  stand  upright. 
(See  Fig.  36.)  To  fill  the  tube,  the  fluid  (it  is  used  only  with 
fluid  media)  is  poured  into  the  bulb  until  this  is  about  half 
full.  The  tube  is  then  tilted  until  the  closed  arm  is  nearly 
horizontal,  so  that  the  air  may  flow  out  into  the  bulb  and  the 
fluid  flow  into  the  closed  arm  to  take  its  place.  When  this 
has  been  completely  filled  sufficient  fluid  should  be  added 
to  bring  its  level  within  the  bulb  just  beyond  the  bend,  and 
the  opening  of  the  bulb  plugged  with  cotton.  The  tubes  thus 
filled  are  then  to  be  sterilized.  During  sterilization  they 
are  to  be  maintained  in  the  upright  position.  Under  the 
influence  of  heat  the  tension  of  the  water-vapor  in  the  closed 
arm  forces  most  of  the  fluid  into  the  bulb.  As  the  tube  cools, 
the  fluid  returns  to  its  place  in  the  closed  arm  and  fills  it 
again,  with  the  exception  of  a  small  space  at  the  top,  which 
is  occupied  by  the  air  originally  dissolved  in  the  liquid  and 

1  An  excellent  and  exhaustive  contribution  to  this  subject  has  been 
made  by  Theobald  Smith  in  the  Wilder  Quarter-Century  Book,  Ithaca, 
N.  Y.,  1803. 


218  BACTERIOLOGY 

which  has  been  driven  out  by  the  heat.  The  air-bubble 
should  be  tilted  out  after  each  sterilization;  and  finally, 
after  the  third  exposure  to  steam,  this  arm  of  the  tube  will 
be  free  from  air.  The  medium  employed  is  bouillon  con- 
taining some  fermentable  carbohydrate,  as  glucose,  lactose, 
or  saccharose.  After  inoculation  the  flasks  are  placed  in  the 
incubator,  and  the  amount  of  gas  that  collects  in  the  closed 

FIG.  36 


Fermentation  tube. 

arm  is  noted  from  day  to  day.  From  studies  that  have  been 
made  this  gas  is  found  to  consist  usually  of  about  one  part 
by  volume  of  carbonic  acid  and  two  parts  by  volume  of  an 
explosive  gas  consisting  largely  of  hydrogen.  For  deter- 
mining the  nature  and  quantitative  relations  of  these  gases 
Smith1  recommends  the  following  procedure:  "The  bulb 

1  Loc.  cit.,  p.  196. 


CHANGES  IN-  THE  REACTION  OF  MEDIA          219 

is  completely  filled  with  a  2  per  cent,  solution  of  sodium 
hydroxide  (NaOH)  and  closed  tightly  with  the  thumb. 
The  fluid  is  shaken  thoroughly  with  the  gas  and  allowed  to 
flow  back  and  forth  from  bulb  to  closed  branch  and  the 
reverse  several  times,  to  insure  intimate  contact  of  the  CO2 
with  the  alkali.  Lastly,  before  removing  the  thumb  all  the 
gas  is  allowed  to  collect  in  the  closed  branch,  so  that  none  may 
escape  when  the  thumb  is  removed.  If  C02  be  present, 
a  partial  vacuum  in  the  closed  branch  causes  the  fluid  to 
rise  suddenly  when  the  thumb  is  removed.  After  allowing 
the  layer  of  foam  to  subside  somewhat  the  space  occupied 
by  gas  is  again  measured,  and  the  difference  between  this 
amount  and  that  measured  before  shaking  with  the  sodium 
hydroxide  solution  gives  the  proportion  of  CO2  absorbed. 
The  explosive  character  of  the  residue  is  determined  as 
follows:  the  thumb  is  placed  again  over  the  mouth  of 
the  bulb  and  the  gas  from  the  closed  branch  is  allowed 
to  flow  into  the  bulb  and  mix  with  the  air  there  present. 
The  plug  is  then  removed  and  a  lighted  match  inserted 
into  the  mouth  of  the  bulb.  The  intensity  of  the  explosion 
varies  with  the  amount  of  air  present  in  the  bulb." 

Durham's  Fermentation  Tube. — Durham  employs  a  con- 
venient modification  of  the  ordinary  fermentation  tube, 
which  is  constructed  in  the  following  manner:  test-tubes 
of  about  10  or  12  c.c.  capacity  are  placed  in  an  inverted 
position  within  a  larger  test-tube,  and  the  latter  plugged 
with  cotton  in  the  usual  way  and  sterilized.  (See  Fig. 
37.)  The  small  tube  should  fit  loosely  within  the  larger 
one.  The  medium  to  be  used  is  run  into  the  larger  tube 
until  there  is  present  about  50  per  cent,  more  than  the 
volume  of  the  smaller  tube.  The  whole  is  then  steri- 
lized in  streaming  steam  by  the  fractional  method.  After 


220 


BACTERIOLOGY 


FIG.  37 


the  first  sterilization  the  small  tube  will  be  found  almost 
filled  with  fluid,  over  which  a  small  air-bubble  lies.  After 
the  second  or  third  sterilization  this 
air-bubble  is  completely  expelled,  and 
the  small  tube  contains  nothing  but 
the  liquid. 

The  medium  that  Durham  employs 
for  the  fermentation  test  is  a  1  per 
cent,  solution  of  Witte's  peptone  in 
distilled  water,  to  which  have  been 
added  known  amounts  of  some  such 
fermentable  sugar  as  glucose,  saccha- 
rose, lactose,  mannite,  etc.,  as  the  case 
may  demand.  He  prefers  peptone  to 
meat-infusion  bouillon  for  the  reason 
that  the  latter  often  contains  traces 
of  muscle-sugar,  and  is  thereby  likely 
to  complicate  the  results.  He  prefers 
neutralization  with  organic  acids  rather 
than  mineral  acids,  and  uses  citric  acid 
by  preference,  the  reason  for  this  being 
that  where  sugars  such  as  those  men- 
tioned are  acted  upon  by  mineral 
acids  under  the  influence  of  heat  their 
composition  is  apt  to  be  altered. 


Durham's   fermentation 
tube. 


NOTE. — Prepare    two   fermentation 
tubes  as  follows:    Fill  one  with  1  per 
cent,   watery  solution  of  peptone  to 
which  2  per  cent,  of  glucose  has  been 
added;  fill  the  other  with  a  similar  peptone  solution,  but 
to  which  only  0.3  per  cent,  of  glucose  has  been  added. 


CHANGES  IN  THE  REACTION  OF  MEDIA          221 

Sterilize  and  inoculate  with  Bacillus  coli  communis.  How 
do  the  two  tubes  differ  from  one  another  after  eighteen 
to  twenty-four  hours  in  the  incubator?  First,  as  regards 
the  reaction  of  the  fluid  in  the  open  arms  of  the  tubes. 
Second,  as  to  accumulation  of  gas  in  closed  arms  of  the 
tubes.  Third,  as  to  the  capacity  of  each  solution  for 
reducing  copper  in  Fehling's  solution.  What  differences 
are  observed,  and  how  may  they  be  explained? 

Indol  Production. — The  detection  of  products  other  than 
those  that  give  rise  to  alterations  in  the  reaction  of  the 
media,  and  whose  presence  may  be  demonstrated  by  chemical 
reactions,  is  a  routine  step  in  the  identification  of  different 
species  of  bacteria.  Among  these  bodies  is  one  that  is  pro- 
duced by  a  number  of  organisms,  and  whose  presence  may 
easily  be  detected  by  its  characteristic  behavior  when 
treated  with  certain  substances.  I  refer  to  nitroso-indol, 
the  reactions  of  which  were  described  by  Beyer  in  1869, 
and  the  presence  of  which  as  a  product  of  the  growth  of 
certain  bacteria  has  since  furnished  a  topic  for  considerable 
discussion. 

Indol,  the  name  by  which  this  body  is  generally  known, 
when  acted  upon  by  reducing  agents  becomes  of  a  more 
or  less  decided  rose  color.  This  body  was  recognized  as 
one  of  the  products  of  growth  of  the  spirillum  of  Asiatic 
cholera  first  by  Poel,  and  a  short  time  subsequently  by 
Bujwid  and  by  Dunham,  and  for  a  time  was  believed  to  be 
peculiarly  characteristic  of  the  growth  of  this  organism. 
It  has  since  been  found  that  there  are  many  other  bacteria 
which  also  possess  the  property  of  producing  indol  in  the 
course  of  their  development.  It  is  constantly  present  in 
putrefying  matters,  and  is  one  of  the  aromatic  compounds 
that  give  to  feces  their  characteristic  odor. 


222  BACTERIOLOGY 

The  methods  employed  for  its  detection  are  as  follows: 
cultivate  the  organism  for  twenty-four  to  forty-eight  hours 
at  a  temperature  of  37°  C.,  in  the  simple  peptone  solution 
known  as  "Dunham's  solution"  (see  formula  for  this 
medium).  This  solution  is  preferred  because  its  pale  color 
does  not  mask  the  rose  color  of  the  reaction  when  the 
amount  of  indol  present  is  very  small. 

Four  tubes  should  always  be  inoculated  and  kept  under 
exactly  the  same  conditions  for  the  same  length  of  .time. 

At  the  end  of  twenty-four  or  forty-eight  hours  the  test 
may  be  made.  Proceed  as  follows:  to  a  tube  containing 
7  c.c.  of  the  peptone  solution,  but  which  has  not  been  inocu- 
lated, add  10  drops  of  concentrated  sulphuric  acid.  To 
another  similar  tube  add  1  c.c.  of  a  0.01  per  cent,  solution 
of  sodium  nitrite,  and  afterward  10  drops  of  concentrated 
sulphuric  acid.  Observe  the  tubes  for  five  to  ten  minutes. 
No  alteration  in  their  color  appears,  or  at  least  there  is  no 
production  of  a  rose  color.  They  contain  no  indol. 

Treat  in  the  same  way,  with  the  acid  alone,  two  of  the 
tubes  which  have  been  inoculated.  If  no  rose  color  appears 
after  five  or  ten  minutes,  add  1  c.c.  of  the  sodium  nitrite 
solution.  If  now  no  rose  color  is  produced,  the  indol  reac- 
tion may  be  considered  as  negative — i.  e.,  no  indol  has  been 
formed  as  a  product  of  the  growth  of  the  bacteria. 

If  indol  is  present,  and  the  rose  color  appears  after  the 
addition  of  the  acid  alone,  it  is  plain  that  not  only  indol 
has  been  formed,  but  coincidently  a  reducing-body.  This  is 
found,  by  proper  means,  to  be  nitrous  acid.  The  sulphuric 
acid  liberates  this  acid  from  its  salts  and  permits  of  its 
reducing  action  being  brought  into  play. 

If  the  rose  color  appears  only  after  the  addition  of  both 
the  acid  and  the  nitrite  solution,  then  indol  has  been  formed 
during  the  growth  of  the  organisms,  but  no  nitrites. 


CHANGES  IN  THE  REACTION  OF  MEDIA          223 

Control  the  results  obtained  by  treating  the  two  remaining 
cultures  in  the  same  way. 

The  test  is  sometimes  made  by  allowing  concentrated 
sulphuric  acid  to  flow  down  the  sides  and  collect  at  the 
bottom  of  the  tube;  the  reaction  is  then  seen  as  a  rose- 
colored  zone  overlying  the  line  of  contact  of  the  acid  and 
culture  medium.  This  method  is  open  to  the  objection  that, 
if  indol  is  present  in  only  a  very  small  amount,  the  faint  rose 
tint  produced  by  it  is  apt  to  be  masked  by  a  brown  color 
that  results  from  the  charring  action  of  the  concentrated 
acid  on  the  other  organic  matters  in  the  culture  medium, 
so  that  its  presence  may  in  this  way  escape  detection.  In 
view  of  this,  Petri  recommends  the  use  of  dilute  sulphuric 
acid.  He  states  that  when  indol  is  present  the  characteristic 
rose  color  appears  a  little  more  slowly  with  the  dilute  acid, 
but  it  is  more  permanent,  and  there  is  never  any  likelihood  of 
its  presence  being  masked  by  other  color  reactions. 

Muir  and  Ritchie  recommend  the  use  of  ordinary  fuming 
or  yellow  nitric  acid  for  this  test.  In  this  method  two  or 
three  drops  of  the  acid  are  added  to  the  culture  under  con- 
sideration. If  indol  be  present,  the  red  color  appears  as  a 
result  of  the  reducing  action  of  the  nitrous  acid  upon  it. 
The  defect  in  this  method  is  that  it  reveals  only  the  presence 
of  indol,  and  fails  to  indicate  whether  or  not  reducing  bodies 
were  coincidently  formed  with  the  indol.  As  a  test  for  indol 
alone  it  is  convenient  and  entirely  trustworthy. 

Reducing  Power  of  Bacteria. — The  power  to  reduce  chemical 
compounds  from  a  higher  to  a  lower  state  may  be  said  to 
be  common  to  all  bacteria.  In  some  bacteria,  perhaps  the 
majority,  it  is  most  conspicuously  manifested  in  connection 
with  substances  containing  sulphur,  hydrogen  sulphide  being 
formed.  In  other  bacteria  it  is  best  seen  in  connection  with 


224  BACTERIOLOGY 

the  alterations  produced  in  certain  pigments,  as  litmus, 
methylene-blue,  indigo,  etc.,  the  normal  color  disappearing 
in  part  or  entirely  according  to  the  nature  and  activity  of 
the  process.  Other  bacteria  have  the  property  of  reducing 
certain  salts,  as  in  the  reduction  of  nitrates  to  nitrites,  or 
even  to  ammonia  by  the  denitrifying  bacteria.  In  some 
instances  these  reductions  result  from  the  fact  that  the 
bacteria  liberate  hydrogen  from  the  compounds,  in  others 
it  results  from  the  fact  that  the  bacteria  abstract  oxygen 
from  such  compounds,  while  in  still  other  instances  the 
reduction  is  of  a  more  complex  nature.  Each  of  these 
changes,  therefore,  indicates  the  nature  of  some  of  the 
metabolic  activities  manifested  by  the  bacteria  in  question. 

Test  for  Hydrogen  Sulphide. — The  reduction  of  sulphur 
compounds  may  be  determined  by  growing  the  bacteria  in 
peptone  solution  containing  ferric  tartrate,  when  the  presence 
of  hydrogen  sulphide  will  be  indicated  by  the  brownish- 
black  or  jet-black  color  of  the  precipitated  iron-sulphide. 

Reduction  of  Nitrates. — The  complete  reduction  of  nitrates 
is  brought  about  by  many  bacteria.  Other  bacteria  are 
capable  of  carrying  the  reducing  action  as  far  as  the  for- 
mation of  ammonia,  while  still  others  merely  reduce  the 
nitrates  to  nitrites.  These  reducing  functions  are  encour- 
aged and  may  be  demonstrated  by  cultivating  the  bacteria 
in  peptone  solution  containing  potassium  nitrate. 

Test  for  Nitrites. — The  method  of  Griess,  as  modified  by 
Ilosvay,  is  quite  satisfactory.  These  reagents  are  required: 

(a)  Naphthylamine 0.1  gram 

Distilled  water 20.0c.c. 

Acetic  acid  (25  per  cent,  solution)    .      .      .  150 . 0  c.c. 

(6)  Sulfanilic  acid 0.5  gram 

Acetic  acid  (25  per  cent,  solution)    .      .      .  150.0  c.c. 


CHANGES  IN  THE  REACTION  OF  MEDIA         225 

In  preparing  solution  a  the  naphthylamine  is  dissolved  in 
20  c.c.  of  boiling  water,  filtered,  allowed  to  cool,  and  mixed 
with  the  dilute  acetic  acid.  Solutions  a  and  b  are  then  mixed. 
It  is  best  prepared  as  needed,  though  it  may  be  preserved 
for  some  time  in  a  glass-stoppered  bottle. 

In  testing  for  nitrites  the  reagent  is  added  in  the  proportion 
of  one  volume  of  reagent  to  five  volumes  of  culture.  When 
nitrites  have  been  formed  a  deep-red  color  appears  in  a  few 
seconds.  If  no  nitrites  have  been  formed  the  culture  remains 
colorless.  In  testing  cultures  it  is  always  necessary  to  control 
the  results  by  blank  tests  on  a  portion  of  the  same  medium 
that  had  not  been  inoculated,  as  some  of  the  ingredients  of 
the  medium  may  have  contained  nitrites. 

Another  test  for  the  formation  of  nitrites  is  a  mixture  of 
starch  and  potassium  iodide,  as  follows: 

Starch 2.0  grams 

Potassium  iodide, 0.5  gram 

Water 100.0  c.c. 

Warm  the  mixture  until  the  starch  is  completely  dis- 
solved. 

In  testing  for  nitrites  add  0.5  c.c.  of  the  reagent  to  a  tube 
of  culture,  and  follow  this  by  the  addition  of  2  or  3  drops 
of  pure  sulphuric  acid.  If  nitrites  have  been  formed,  a 
dark-blue  or  purple  color  will  appear.  Control-tubes  of  the 
medium  show  no  color  reaction,  or  merely  a  trace  of  blue 
coloration. 

Test  for  Ammonia. — The  formation  of  ammonia  may  be 
detected  by  testing  with  Nessler's  reagent.  The  most  satis- 
factory results  are  obtained  by  cultivating  the  organisms 
in  a  liter  of  culture  fluid  and  then  distilling  off  portions  of 
the  culture,  collecting  in  Nessler  tubes,  and  applying  1  c.c. 
of  the  reagent  to  each  50  c.c.  of  the  distillate.  The  presence 
15 


226  BACTERIOLOGY 

of  ammonia  in  the  distillate  is  shown  by  the  yellow  coloration 
resulting  from  the  addition  of  the  reagent. 

The  direct  application  of  the  reagent  to  the  culture  will 
give  satisfactory  results  if  a  great  deal  of  ammonia  has  been 
formed.  In  this  instance  the  mercury  in  the  reagent  will 
be  precipitated  as  mercurous  oxide.  Another  rough  test  for 
the  formation  of  ammonia  is  to  place  a  strip  of  filter-paper — 
moistened  with  the  Nessler  reagent — over  the  mouth  of  a 
test-tube  containing  the  culture,  and  then  gently  heating 
the  culture.  As  the  ammonia  is  driven  off  by  the  heat,  it 
will  react  on  the  reagent  on  the  strip  of  paper. 

Examination  of  Cultures  for  Bacterial  Toxins.— In  the  sys- 
tematic study  of  a  pathogenic  organism  it  is  necessary  to 
know  whether  it  is  capable  of  producing  a  soluble  toxin 
when  growing  in  culture  media.  This  is  done  by  filtering 
cultures  of  various  ages  and  testing  the  effect  of  the  filtrate 
upon  susceptible  animals. 

FILTRATION  OF  CULTURES. — A  variety  of  filters  have  been 
devised  for  the  purpose  of  filtering  liquid  cultures  and  other 
fluids  to  obtain  sterile  filtrates.  These  filters  are  usually 
constructed  of  unglazed  porcelain  or  of  infusorial  earth,  and 
are  made  in  the  form  of  hollow  cylinders  or  bulbs.  The  best- 
known  forms  of  bacterial  filters  are  the  Chamberland  and 
the  Berkefeld.  All  the  filters  used  for  this  purpose  require 
some  motive  power  to  force  the  fluid  through  the  filter. 
Compressed  air  may  be  employed  to  force  the  fluid  through 
the  filter,  or  atmospheric  pressure  may  be  utilized  by  creating 
a  negative  pressure  on  the  distal  side  of  the  filter  by  the  use 
of  an  air-pump. 

It  is  always  necessary  to  test  the  sterility  of  the  filtrate 
by  making  cultures  from  it  into  nutritive  media  and  noting 
whether  growth  takes  place  or  not. 


ANAEROBIC  METHODS  227 

Cultivation  without  Oxygen. — As  we  have  already  learned, 
there  is  a  group  of  bacteria  to  which  the  designation  "anae- 
robic" has  been  given,  which  are  characterized  by  inability 
to  grow  in  the  presence  of  free  oxygen.  For  the  cultivation 
of  the  members  of  this  group,  a  number  of  devices  are 
employed  for  the  exclusion  of  free  oxygen  from  the  cultures. 

Method  of  Buchner.  The  plan  suggested  by  Buchner,  of 
allowing  the  cultures  to  develop  in  an  atmosphere  robbed  of 
its  oxygen  by  pyrogallic  acid,  gives  very  good  results.  In 
this  method  the  culture,  which  is  either  a  slant-  or  stab- 
culture  in  a  test-tube,  is  placed — tube,  cotton  plug,  and  all — 
into  a  larger  tube,  in  the  bottom  of  which  have  been  deposited 
1  gram  of  pyrogallic  acid  and  10  c.c.  of  yV  normal  caustic- 
potash  solution.  The  larger  tube  is  then  tightly  plugged 
with  a  rubber  stopper.  The  oxygen  is  quickly  absorbed 
by  the  pyrogallic  acid,  and  the  organisms  develop  in  the 
remaining  constituents  of  the  atmosphere,  viz.,  nitrogen,  a 
small  amount  of  CO2,  and  a  trace  of  ammonia. 

Method  of  C.  Frdnkel.  Carl  Frankel  suggested  the  fol- 
lowing :  the  tube  is  first  inoculated  as  if  it  were  to  be  poured 
as  a  plate  or  rolled  as  an  ordinary  Esmarch  tube.  The  cotton 
plug  is  then  replaced  by  a  rubber  stopper,  through  which 
pass  two  glass  tubes.  These  must  all  have  been  sterilized 
in  the  steam  sterilizer  before  using.  On  the  outer  side  of 
the  stopper  these  two  tubes  are  bent  at  right  angles  to  the 
long  axis  of  the  test-tube  into  which  they  are  to  be  placed, 
and  both  are  slightly  drawn  out  in  a  gas-flame.  Both  of 
these  tubes  must  be  provided,  before  sterilization,  with 
a  plug  of  cotton;  this  is  to  prevent  the  access  of  foreign 
organisms  to  the  medium  during  manipulations.  At  the 
inner  side  of  the  rubber  stopper — that  is,  the  end  which  is 
to  be  inserted  into  the  test-tube — the  glass  tubes  are  of 


228 


BACTERIOLOGY 


different  lengths:  one  reaches  to  within  0.5  cm.  of  the  bottom 
of  the  test-tube,  the  other  is  cut  off  flush  with  the  under 
surface  of  the  stopper.  The  outer  end  of  the  longer  glass 
tube  is  then  connected  with  a  hydrogen  generator  and 
hydrogen  is  allowed  to  bubble  through  the  gelatin  (Fig.  38,  A) 

FIG.  38 


Franke!' s  method  for  the  cultivation  of  anaerobic  bacteria. 


in  the  tube  until  all  contained  air  has  been  expelled  and 
.its  place  taken  by  the  hydrogen.1    When  the  hydrogen  has 

1  Before  beginning  the  experiment  it  is  always  wise  to  test  the  hydro- 
gen— i.  e.,  to  see  that  it  is  free  from  oxygen  and  that  there  is  no  danger 
of  an  explosion,  for  unless  this  be  done  the  entire  apparatus  may  be  blown 
to  pieces  and  a  serious  accident  occur.  The  agents  used  should  be  pure 
zinc  and  pure  sulphuric  acid  of  about  25  to  30  per  cent,  strength.  With  the 
primary  evolution  of  the  gas  the  outlet  of  the  generator  should  be  closed 
and  kept  closed  until  the  gas  reservoir  is  quite  filled  with  hydrogen.  The 


ANAEROBIC  METHODS  229 

been  bubbling  through  the  gelatin  for  about  five  minutes 
(at  least)  one  can  be  reasonably  sure  that  all  oxygen  has 
been  expelled.  The  drawn-out  portions  of  the  tubes  can 
then  be  sealed  in  the  gas-flame  without  fear  of  an  explosion. 
The  protruding  end  of  the  rubber  stopper  is  then  painted 
around  with  melted  paraffin  and  the  tube  rolled  in  the  way 
given  for  ordinary  Esmarch  tubes.  A  tube  thus  prepared 
and  containing  growing  colonies  is  shown  in  Fig.  38,  B. 

The  development  that  now  occurs  is  in  an  atmosphere  of 
hydrogen,  all  oxygen  having  been  expelled.  During  the 
operation  the  tube  containing  the  liquefied  gelatin  should 
be  kept  in  a  water-bath  at  a  temperature  sufficiently  high 
to  prevent  its  solidifying,  and  at  the  same  time  not  high 
enough  to  kill  the  organisms  with  which  it  has  been  inocu- 
lated. 

One  of  the  obstacles  to  the  successful  performance  of  this 
method  is  the  bubbling  of  the  gelatin,  the  foam  from  which 
will  often  fill  the  exit-tube  and  sometimes  be  forced  from  it. 
This  may  be  obviated  by  reversing  the  order  of  proceeding, 
viz.:  roll  the  Esmarch  tube  in  the  ordinary  way  with  the 
organisms  to  be  studied,  using  a  relatively  small  amount  of 
gelatin,  so  as  to  have  as  thin  a  layer  as  possible  when  it  is 
rolled.  Then  replace  the  cotton  plug  with  the  sterilized 

outlet  should  then  be  opened  and  the  entire  volume  of  gas  allowed  to  escape, 
care  being  taken  that  no  flame  is  in  the  neighborhood.  This  should  be 
repeated,  after  which  a  sample  of  the  hydrogen  generated  should  be  collected 
in  an  inverted  test-tube  in  the  ordinary  way  for  collecting  gases  over  water, , 
viz.,  by  filling  a  test-tube  with  water,  closing  its  mouth  with  the  thumb, 
inverting  it,  and  placing  its  mouth  under  water,  when,  after  removing  the 
thumb,  the  water  will  be  kept  in  it  by  atmospheric  pressure.  The  hydrogen 
which  is  flowing  from  the  open  generator  may  be  conducted  to  the  test- 
tube  by  rubber  tubing.  When  the  water  has  been  replaced  test  the  gas 
by  holding  a  flame  near  the  open  mouth  of  the  test-tube.  If  no  explo- 
sion occurs,  the  hydrogen  is  safe  to  use.  Should  there  be  an  explosion,  the 
generation  of  hydrogen  must  be  continued  in  the  apparatus  until  it  burns 
with  a  colorless  flame  when  tested  in  a  test-tube. 


230  BACTERIOLOGY 

rubber  stopper  carrying  the  glass  tubes  through  which  the 
hydrogen  is  to  be  passed,  and  allow  the  hydrogen  to  flow 
through  as  in  the  method  first  given.  The  gas  now  passes 
over  the  gelatin  instead  of  through  it,  and  consequently  no 
bubbling  results.  In  all  other  respects  the  procedure  is  the 
same  as  that  given  by  Frankel. 

Method  of  Kitasato  and  Weil. — For  favoring  anaerobic 
conditions  Kitasato  and  Weil  have  suggested  the  addition 
to  the  culture  media  of  some  strong  reducing-agent.  They 
recommend  formic  acid  or  sodium  formate,  in  0.3  to  0.5  per 
cent.;  glucose  in  1.5  to  2  per  cent.;  or  blue  litmus  tincture 
in  5  per  cent,  by  volume.  This  is,  of  course,  in  addition  to 
an  atmosphere  from  which  all  oxygen  has  been  expelled. 
As  a  reducing  agent  for  this  purpose,  Theobald  Smith  regards 
a  weaker  solution  of  glucose,  0.3  to  0.5  per  cent.,  as  more 
advantageous;  and  Wright  obtains  better  results  when 
glucose  is  added  if  the  primary  reaction  of  the  media  is  about 
neutral  to  phenolphthalein. 

Method  of  Park.  A  very  simple,  convenient,  and  effi- 
cient method  is  employed  by  Park.  It  consists  in  covering 
the  medium  in  which  the  anaerobic  species  are  to  be  cul- 
tivated with  liquid  paraffin  (albolene).  The  best  results 
are  obtained  when  the  amount  of  paraffin  added  is  about 
half  that  of  the  liquid  in  the  tube  or  flask.  The  liquid  paraffin 
has  the  advantage  over  the  solid  paraffin  in  not  retracting 
from  the  walls  of  the  vessel  on  cooling.  All  air  is  expelled 
from  flasks  or  tubes  prepared  in  this  way,  by  heating  them 
in  the  autoclave.  The  layer  of  paraffin  prevents  the  reab- 
sorption  of  oxygen  driven  off  by  the  heat.  After  cooling,  the 
inoculation  is  made  by  passing  the  needle  through  the  paraffin 
well  down  into  the  media. 

Many  other  methods  are  employed  for  this  special  purpose, 
but  for  the  beginner  those  given  will  suffice. 


ANAEROBIC  METHODS  231 

From  what  has  been  said,  it  may  be  inferred  that  the  cul- 
tivation of  anaerobic  bacteria  is  a  simple  matter  attended 
with  but  little  difficulty.  Such  an  opinion  will,  however, 
be  quickly  abandoned  when  the  beginner  attempts  this  part 
of  his  work  for  the  first  time,  and  particularly  when  his 
efforts  are  directed  toward  the  separation  of  these  forms  from 
other  organisms  with  which  they  are  associated.  The 
presence  of  spore-forming,  facultative  anaerobes  in  mixed 
cultures  is  always  to  be  suspected,  and  it  is  this  group  that 
renders  the  task  so  difficult.  At  best  the  work  requires  undi- 
vided attention  and  no  small  degree  of  skill  in  bacteriological 
technique. 


CHAPTER  XII. 

Inoculation  of  Animals — Subcutaneous  Inoculation — Intravenous  Injec- 
tion— Inoculation  into  the  Lymphatic  Circulation — Inoculation  into 
the  Great  Serous  Cavities,  and  into  the  Anterior  Chamber  of  the  Eye- 
Observation  of  Animals  after  Inoculation. 


AFTER  subjecting  an  organism  to  the  methods  of  study 
that  we  have  thus  far  reviewed  there  remains  to  be  tested 
its  action  on  animals — i.  e.,  to  determine  if  it  possesses  the 
property  of  producing  disease  or  not;  and,  if  so,  what  are 
the  pathological  results  of  its  growth  in  the  tissues  of  animals, 
and  in  what  way  must  it  gain  entrance  to  the  tissues  in 
order  to  produce  those  results?  The  mode  of  deciding  these 
points  is  by  inoculation,  which  is  practised  in  different  ways 
according  to  circumstances.  Most  commonly  a  bit  of  the 
culture  to  be  tested  is  simply  deposited  beneath  the  skin  of 
the  animal;  but  in  other  cases  it  may  be  necessary  to  intro- 
duce it  directly  into  the  vascular  or  lymphatic  circulation, 
or  into  one  or  the  other  of  the  great  serous  cavities;  or, 
for  still  other  purposes  of  observation,  into  the  anterior 
chamber  of  the  eye,  upon  the  iris  or  within  the  skull  cavity, 
upon  the  dura  or  brain  substance. 

SUBCUTANEOUS   INOCULATION   OF   ANIMALS. 

The  animals  usually  employed  in  the  laboratory  for  pur- 
poses of  inoculation  are  white  mice,  gray  house-mice,  guinea- 
pigs,  rabbits,  and  pigeons. 

For  simple  subcutaneous  inoculation  the  steps  in  the 
(232) 


SUBCUTANEOUS  INOCULATION  OF  ANIMALS     233 

process  are  practically  the  same  in  all  cases.  The  hair  or 
feathers  are  to  be  carefully  removed.  If  the  skin  is  very 
dirty,  it  may  be  scrubbed  with  soap  and  water.  Sterili- 
zation of  the  skin  is  practically  impossible,  so  it  need  not  be 
attempted.  If  the  inoculation  is  to  be  made  by  means  of  a 
hypodermic  syringe,  then  a  fold  of  the  skin  may  be  lifted 
up  and  the  needle  inserted  in  the  usual  way.  If  a  solid 
culture  is  to  be  inoculated,  a  fold  of  skin  may  be  taken  up 
with  forceps  and  a  tiny  pocket  cut  into  it  with  scissors 
which  have  previously  been  sterilized.  This  pocket  must 
be  large  enough  to  admit  the  end  of  the  needle  without  its 
touching  the  sides  of  the  opening  as  it  is  inserted.  Beneath 
the  skin  will  be  found  the  superficial  and  deep  connective- 
tissue  fascise.  These  must  be  taken  up  with  sterilized 
forceps,  and  with  sterilized  scissors  incised  in  a  way  corre- 
sponding to  the  opening  in  the  skin.  The  pocket  is  then  to 
be  held  open  with  the  forceps  and  the  substance  to  be 
inserted  is  introduced  as  far  under  the  skin  and  fasciae  as 
possible,  care  being  taken  not  to  touch  the  edges  of  the 
wound  if  it  can  be  avoided.  The  edges  of  the  wound  may 
then  be  simply  pulled  together  and  allowed  to  remain.  No 
stitching  or  efforts  at  closing  it  are  necessary,  though  a  drop 
of  collodion  over  the  point  of  operation  may  serve  to  lessen 
contamination. 

As  the  subcutaneous  inoculation  is  very  simple  and  takes 
only  a  few  moments,  guinea-pigs,  rabbits,  and  pigeons  may 
be  held  by  an  assistant.  The  front  legs  in  the  one  hand 
and  the  hind  legs  in  the  other,  with  the  animal  stretched 
upon  its  back  on  a  table,  is  the  usual  position  for  the  opera- 
tion when  practised  upon  guinea-pigs  and  rabbits.  The 
point  at  which  the  inoculations  are  commonly  made  is  in 
the  abdominal  wall,  either  to  the  right  or  left  of  the  median 


234  BACTERIOLOGY 

line  and  about  3  cm.  distant.  When  pigeons  are  used  they 
are  held  with  the  legs,  tail,  and  ends  of  the  wings  in  the  one 
hand,  and  the  head  and  anterior  portion  of  the  body  in  the 
other,  leaving  the  area  occupied  by  the  pectoral  muscles, 
over  which  the  inoculation  is  to  be  made,  free  for  manipu- 
lation. In  the  case  of  fur-bearing  animals  the  hair  over  the 
point  selected  for  the  inoculation  should  be  closely  cut  with 
scissors,  and  from  a  small  area  the  feathers  should  be  plucked 
in  the  case  of  birds. 

FIG.  39 


Kitasato's  mouse-holder. 

It  is  at  times,  however,  more  convenient  to  dispense  with 
an  assistant;  one  of  several  forms  of  apparatus  that  have 
been  devised  for  holding  mice,  guinea-pigs,  rats,  rabbits, 
etc.,  may  then  be  used.  For  small  animals,  such  as  mice  and 
rats,  the  holder  suggested  by  Kitasato  is  very  useful.  It 
is  simply  a  metal  plate  attached  to  a  stand  by  a  clamped 
ball-and-socket  joint,  so  that  it  can  be  fixed  in  any  position. 
It  is  provided  with  a  spring-clip  at  one  end  that  holds  the 


SUBCUTANEOUS  INOCULATION  OF  ANIMALS    235 

animal  by  the  skin  of  the  neck,  and  at  the  other  end  with 
another  clamp  that  holds  the  tail  of  the  animal.  This 
holder  is  shown  in  Fig.  39.  For  larger  animals  the  form  of 
holder  shown  in  Fig.  40  is  commonly  used. 

The  holder  devised  by  Sweet,1  which  can  be  made  of  any 
size,  from  that  suitable  to  a  guinea-pig  up  to  that  large 
enough  to  secure  a  dog,  is  in  every  way  the  most  convenient 
that  we  have  encountered  and,  from  the  standpoint  of  the 
animal,  is  the  most  humane.  It  consists  of  four  pieces  of 
heavy  round  wire  so  bent  that  two  engage  the  animal 

FIG.  40 


Holder  for  larger  animals. 

immediately  behind  the  lower  jaw  while  the  remaining  two 
close  over  the  muzzle.  All  are  held  in  position  by  a  single 
clamp  controlled  Jby  a  single  thumb-screw.  When  the  screw 
is  reversed  and  the  clamp  opened  the  anterior  and  posterior 
wire  of  each  pair  falls  away  from  the  median  line,  thereby 
liberating  the  animal.  To  secure  the  animal  it  is  placed 
upon  its  back,  the  head  laid  in  the  cradle  formed  by  the 
bent  wires,  the  latter  are  adjusted  to  the  proper  position, 

1A   Simple,    Humane   Holder   for   Small   Animals    under    Experiment, 
University  of  Penna.  Med.  Bull.,  1903,  No.  2,  p.  78. 


236 


BACTERIOLOGY 


and  all  secured  by  the  turn  of  the  single  set-screw.  Of 
course,  the  extremities  of  the  animal  are  to  be  secured.  This 
is  done  by  means  of  cords  securely  held  by  a  patent  fastener 
made  by  the  Tie  Co.,  of  Unadilla,  N.  Y.  These  fasteners 
are  in  every  way  more  convenient  than  the  cleats  in  common 
use.  An  idea  of  the  apparatus  is  given  in  Fig.  41. 


FIG.  41 


A  very  simple  and  useful  holder  for  guinea-pigs  consists 
of  a  metal  cylinder  of  about  5  cm.  diameter  and  about  13 
cm.  long,  closed  at  one  end  by  a  perforated  cap  of  either  tin 
or  wire  netting.  Along  the  side  of  this  box  is  a  longitudinal 
slit  12  mm.  wide  that  runs  for  9.5  cm.  from  within  0.5  cm. 
of  the  open  extremity  of  the  cylinder.  The  animal  is  placed 


SUBCUTANEOUS  INOCULATION  OF  ANIMALS     237 

in  such  a  cylinder  with  its  head  toward  the  perforated 
bottom.  It  is  then  easily  possible  to  make  subcutaneous 
inoculation  by  taking  up  a  bit  of  skin  through  the  slit  in  the 


FIG.  42 


The  Voges  holder  for  guinea-pigs. 


side  of  the  box,  or  to  make  intraperitoneal  injection  by  draw- 
ing the  posterior  extremities  slightly  from  the  box  and  hold- 
ing them  steady  between  the  index  and  second  finger,  as 
seen  in  Fig.  42.  It  is  also  very  convenient  for  use  when  the 


238  BACTERIOLOGY 

rectal  temperature  of  these  small  animals  is  to  be  taken. 
The  manipulation  can  easily  be  made  without  the  aid  of 
an  assistant.  Its  construction  is  seen  in  Fig.  42. 1 

For  ordinary  subcutaneous  inoculations  at  the  root  of  the 
tail  in  mice  a  simple  apparatus  consists  of  a  piece  of  board 
about  7  x  10  cm.  and  2  cm.  thick,  upon  which  is  tacked  a 
hollow  truncated  cone  of  wire  gauze,  about  6  cm.  long  and 
about  1.5  cm.  in  diameter  at  one  end  and  2  cm.  at  its  other 
end.  This  is  tacked  upon  the  board  in  such  a  position  that 
its  long  axis  is  in  the  long  axis  of  the  board,  being  equidistant 
from  its  sides.  Its  small  end  is  placed  at  the  edge  of  the 

FIG.  43 


Mouse-holder,  with  mouse  in  proper  position. 

board.  .The  mouse  is  taken  up  by  the  tail  by  means  of  a 
pair  of  tongs  and  allowed  to  crawl  into  the  smaller  end  of 
the  wire  cone.  When  so  far  in  that  only  the  root  of  the 
tail  projects  the  animal  is  fixed  in  this  position  by  a  clamp 
and  thumb-screw,  with  which  the  apparatus  (Fig.  43)  is 
provided.  The  animal  usually  remains  perfectly  quiet  and 
may  be  handled  without  difficulty. 

The  hair  over  the  root  of  the  tail  is  to  be  carefully  cut 
away  with  scissors  and  a  pocket  cut  through  the  skin  at 
this  point.  The  inoculation  is  then  made  into  the  loose 

1  Centralblatt  fur  Bacteriologie  and  Parasitenkunde,  1895,  vol.  xviii,  p.  530. 


•SUBCUTANEQUS  INOCULATION  OF  ANIMALS      239 

tissue  under  the  skin  over  this  part  of  the  back  in  the  way 
that  has  just  been  described.  It  is  always  best  to  insert  the 
needle  some  distance  along  the  spinal  column,  and  thus 
deposit  the  material  as  far  from  the  surface-wound  as 
possible. 

Injection  into  the  Circulation. — It  is  not  infrequently 
desirable  to  inject  the  material  under  consideration  directly 
into  the  circulation  of  an  animal.  If  a  rabbit  is  employed 
for  the  purpose,  the  operation  is  usually  done  upon  one  of 
the  veins  in  the  ear.  To  those  who  have  had  no  practice 
with  this  procedure  it  offers  a  great  many  difficulties;  but 
if  the  directions  which  will  be  given  are  strictly  observed 
the  greatest  of  these  obstacles  to  the  successful  performance 
of  the  operation  may  be  overcome. 

When  viewing  the  circulation  in  the  ear  of  the  rabbit  by 
transmitted  light  three  conspicuous  branches  of  the  main 
vessel  (vena  auricularis  posterior)  will  be  seen.  One  runs 
about  centrally  in  the  long  axis  of  the  ear,  one  runs  along 
its  anterior  margin,  and  one  along  its  posterior  margin. 
The  central  branch  (ramus  anterior  of  the  vena  auricularis 
posterior)  is  the  largest  and  most  conspicuous  vessel  of  the 
ear,  and  is,  therefore,  believed  by  the  inexperienced  to  be 
the  branch  into  which  it  would  appear  easiest  to  insert  a 
hypodermic  needle.  This,  however,  is  fallacious.  This 
vessel  lies  very  loosely  imbedded  in  connective  tissue,  and, 
in  efforts  to  introduce  a  needle  into  it,  rolls  about  to  such 
an  extent  that  only  after  a  great  deal  of  difficulty  does  the 
experiment  succeed.  On  the  other  hand,  the  posterior 
branch  (ramus  lateralis  posterior  of  the  vena  auricularis 
posterior)  is  a  very  fine,  delicate  vessel  which  runs  along  the 
posterior  margin  of  the  ear,  and  is  so  firmly  fixed  in  the  dense 
tissues  which  surround  it  that  it  is  prevented  from  rolling 


240  BACTERIOLOGY 

about  under  the  point  of  the  needle.  The  further  away  from 
the  mouth  of  the  vessel — that  is,  the  nearer  we  approach 
its  capillary  extremity — the  more  favorable  become  the 
conditions  for  the  success  of  the  operation. 

After  shaving  the  ear  and  carefully  washing  it  with  clean 
water  select  the  very  delicate  vessel  lying  quite  close  to  the 
posterior  margin  of  the  ear,  and  make  the  injection  as  near 
to  the  apex  of  the  ear  as  possible.  At  times  the  vessels  of 
the  ear  will  be  found  to  contain  so  little  blood  that  they  are 
hardly  distinguishable,  making  it  very  difficult  to  introduce 
the  needle  into  them.  This  is  sometimes  overcome  by  pres- 
sure at  the  root  of  the  ear,  causing  stasis  of  the  blood  and 
distention  of  the  vessels.  A  very  satisfactory  method  of 
causing  the  veins  to  become  prominent  is  to  press  lightly  or 
prick  gently  with  the  point  of  a  needle  the  skin  over  the 
vessel  to  be  used.  In  a  few  seconds,  as  a  result  of  this  irri- 
tation, the  vessel  will  have  become  distended  with  blood, 
and  readily  distinguishable  from  the  surrounding  tissue; 
it  may  then  be  easily  punctured  by  the  needle  of  the  syringe. 
A  sharp  flick  with  the  finger  will  often  produce  the  same 
result.  The  injection  is  always  to  be  made  from  the  dorsal, 
surface  of  the  ear. 

Of  no  less  importance  than  the  selection  of  the  proper 
vessel  is  the  shape  of  the  point  of  the  needle  employed. 
The  hypodermic  needles  as  they  come  from  the  makers 
are  not  suited  at  all  for  this  operation,  because  of  the  manner 
in  which  their  points  are  ground.  If  one  examine  carefully 
the  point  of  a  new  hypodermic  needle,  it  will  be  seen  that 
the  long  point,  instead  of  presenting  a  flat,  slanting  surface 
when  viewed  from  the  side,  has  a  more  or  less  curved  surface. 
Now,  in  efforts  to  introduce  such  a  needle  into  a  vessel  of 
very  small  caliber  it  is-  usually  seen  that  the  point  of  the 


SUBCUTANEOUS  INOCULATION  OF  ANIMALS     241 

needle,  instead  of  remaining  in  the  vessel,  as  it  would  do 
were  it  straight  (or  "chisel  pointed"),  very  commonly  pro- 
jects into  the  opposite  wall;  and  as  the  needle  is  inserted 
further  and  further  it  is  usually  pushed  through  the  vessel- 
walls  into  the  loose  tissues  beyond,  and  the  material  to  be 
injected  is  deposited  in  these  tissues,  instead  of  into  the 
circulation.  If,  on  the  contrary,  the  slanting  point  of  the 
needle  be  ground  until  its  surface  is  perfectly  flat  when 
viewed  from  the  side,  and  no  curvature  exists,  then  when 
once  inserted  it  usually  remains  within  the  vessel,  and  there 


FIG.  44 
a 


Hypodermic  needles,  magnified,     a,  improper  point;    6,  proper  shape  of 

point. 


is  no  tendency  to  penetrate  the  opposite  wall.  We  never 
use  a  new  hypodermic  needle  until  its  point  is  carefully 
ground  to  a  perfectly  flat,  slanting  surface  with  no  curvature 
whatever. 

These  differences  may  perhaps  be  more  easily  understood 
if  represented  diagrammatically.  In  Fig.  44,  a,  the  needle 
has  the  point  usually  seen  when  new.  In  Fig.  44,  b,  the 
point  has  been  ground  to  the  shape  best  suited  for  this 
operation.  The  needles  need  not  be  returned  to  the  maker. 
One  can  grind  them  to  the  shape  desired  in  a  few  minutes 
upon  an  oilstone.  The  size  of  the  needle  is  that  commonly 
16 


242  BACTERIOLOGY 

employed  by  physicians  for  subcutaneous  injections  in 
human  beings. 

When  the  operation  is  to  be  performed  an  assistant  holds 
the  animal  gently  but  firmly  in  the  crouching  position  upon 
a  table.  If  the  animal  does  not  remain  quiet,  it  is  best  to 
wrap  it  in  a  towel,  so  that  only  its  head  protrudes;  though 
in  most  cases  we  have  not  found  this  necessary,  particularly 
if  the  animal  has  not  been  excited  prior  to  beginning  the 
operation. 

The  ear  in  which  the  injection  is  to  be  made  should  be 
shaved  clean  of  hair  by  means  of  a  razor  and  soap  and  then 
washed  with  water.  It  is  unnecessary  to  attempt  disin- 
fection of  the  skin. 

The  animal  should  be  placed  so  that  the  prepared  ear 
comes  between  the  operator  and  the  source  of  light.  This 
renders  visible  by  transmitted  light  not  only  the  coarser 
vessels  of  the  ear,  but  also  their  finer  branches. 

The  filled  hypodermic  syringe  is.  taken  in  one  hand  and 
with  the  other  hand  the  ear  is  held  firmly.  The  point  of  the 
needle  is  then  inserted  through  the  skin  and  into  the  finest 
part  of  the  ramus  posterior,  the  part  nearest  the  apex  of  the 
ear,  where  the  course  of  the  vessel  is  nearly  straight.  When 
the  point  of  the  needle  is  in  this  vessel  it  gives  to  the  hand 
a  sensation  quite  different  from  that  felt  when  it  is  in  the 
midst  of  connective  tissue.  As  soon  as  one  supposes  the 
point  of  the  needle  is  in  the  vessel  a  drop  or  two  of  the  fluid 
may  be  injected  from  the  syringe,  and,  if  his  suspicions 
are  correct,  the  circulation  in  the  small  ramifications  and 
their  anastomoses  will  rapidly  alter  in  appearance — i.  e., 
the  circulating  blood  will  be  displaced  very  quickly  by  the 
clear,  transparent  fluid  that  is  being  injected.  At  this  stage 
one  must  proceed  very  carefully,  for  sometimes  when  the 


SUBCUTANEOUS  INOCULATION  OF  ANIMALS     243 

needle-point  is  not  actually  in  the  vessel,  but  is  in  the  lymph 
spaces  surrounding  it,  an  appearance  somewhat  similar  is 
seen.  This  may  always  be  differentiated,  however,  by  con- 
tinuing the  injection,  when  the  flow  of  clear  fluid  through 
the  vessels  will  not  only  fail  to  take  the  place  of  the  cir- 
culating blood,  but  at  the  same  time  a  localized  swelling, 
due  to  an  accumulation  of  the  fluid  injected,  will  appear 
under  the  skin  about  the  point  of  the  needle.  The  needle 
must  then  be  withdrawn  and  inserted  into  the  vessel  at  a 
point  a  little  nearer  its  proximal  end. 

Care  must  be  taken  that  no  air  is  injected. 

The  hypodermic  syringe  and  needle  must,  previous  to 
operation,  have  been  carefully  sterilized  in  the  steam  steril- 
izer or  in  boiling  water.  The  animal  must  be  kept  under 
close  observation  for  about  an  hour  after  injection. 

The  operation  is  one  that  cannot  be  learned  from  verbal 
description.  It  can  only  be  successfully  performed  after 
actual  practice.  If  the  precautions  which  have  been  men- 
tioned are  observed,  but  little  difficulty  in  performing  the 
operation  will  be  experienced. 

Its  greater  convenience  and  simplicity,  as  compared  with 
other  methods  for  the  introduction  of  substances  into  the 
circulation,  commend  it  as  a  technical  procedure  with  which 
to  make  one's  self  familiar.  The  animals  sustain  practically 
no  wound,  they  experience  no  suffering — at  least  they  give 
no  evidence  of  pain — and  no  anesthetic  is  required. 

The  form  of  syringe  best  suited  for  this  operation  is  of 
the  ordinary  design,  but  one  that  permits  of  thorough 
sterilization  by  steam.  It  should  be  made  of  glass  and  metal, 
with  packings  that  may  be  sterilized  by  steam  without 
injury.  The  syringes  commonly  employed  are  those  shown 
in  Fig.  45. 


244 


BACTERIOLOGY 


For  operations  requiring  exact  dosage  experience  has  led 
me  to  prefer  a  syringe  after  the  pattern  of  C,  in  Fig.  45 — i.  e., 
the  form  commonly  used  by  physicians.  The  reason  for 
this  is  as  follows:  in  making  injections,,  either  into  the  cir- 
culation or  under  the  skin,  there  is  a  certain  amount  of 
resistance  to  the  passage  of  fluid  from  the  needle.  If  one 
overcomes  this  resistance  by  means  of  a  cushion  of  com- 
pressed air,  as  is  the  case  in  syringes  A  and  B,  Fig.  45,  the 
sudden  expansion  of  the  air  in  the  body  of  the  syringe  when 

FIG.  45 


Forms  of  hypodermic  syringe.    A,  Koch's  syringe;  B,  syringe  of  Strohschein; 
C,  Overlack's  form. 

resistance  is  overcome  frequently  causes  a  larger  amount 
of  fluid  to  be  injected  than  is  desired.  No  such  accident 
is  likely  to  occur  when  the  fluid  is  forced  from  the  barrel 
of  the  syringe  by  the  head  of  a  close-fitting  piston,  with  no 
air  intervening  between  the  fluid  and  the  head  of  the  piston. 
With  such  an  instrument,  properly  manipulated,  the  dose 
can  always  be  controlled  with  accuracy. 

Inoculation  into  the  Lymphatic  Circulation. — Fluid  cultures 
or  suspensions  of  bacteria  may  be  injected  into  the  lym- 


SUBCUTANEOUS  INOCULATION  OF  ANIMALS     245 

phatics  by  way  of  the  testicles.  The  operation  is  in  no  wise 
complicated.  One  simply  plunges  the  point  of  the  hypo- 
dermic needle  directly  into  the  substance  of  the  testicle  and 
then  injects  the  amount  desired.  Injections  made  in  this 
manner  are  usually  followed  by  instructive  pathological 
lesions  of  the  lymphatic  apparatus  of  the  abdomen. 

Inoculation  into  the  Great  Serous  Cavities. — Inoculation  into 
the  peritoneum  presents  no  difficulties  if  fluids  are  to  be 
introduced.  In  this  case  one  makes,  with  a  pair  of  sterilized 
scissors,  a  small  nick  through  the  skin  down  to  the  under- 
lying fasciae,  and,  taking  a  fold  of  the  abdominal  wall  between 
the  fingers,  plunges  the  hypodermic  needle  through  the 
opening  just  made  directly  into  the  peritoneal  cavity.  There 
is  little  or  no  danger  of  penetrating  the  intestines  or  other 
internal  viscera  if  the  puncture  be  made  along  the  median 
line  at  about  midway  between  the  end  of  the  sternum 
and  the  symphysis  pubis.  Though  this  may  seem  a  rude 
method  it  is  rare  that  the  intestines  are  penetrated  or  other- 
wise injured.  The  object  of  the  primary  incision  is  to  lessen 
the  chances  of  contamination  by  bacteria  located  in  the  skin, 
some  of  which  might  adhere  to  the  needle  if  it  were  plunged 
directly  through  the  skin,  and  thus  complicate  the  results. 

If  solid  substances,  bits  of  tissue,  etc.,  are  to  be  intro- 
duced into  the  peritoneum,  it  becomes  necessary  to  conduct 
the  operation  under  an  anesthetic  and  upon  the  lines  of  a 
laparotomy.  The  hair  should  be  shaved  from  a  small  area 
over  the  median  line,  after  which  the  skin  is  to  be  thoroughly 
washed..  A  short  longitudinal  incision  (about  2  cm.  long) 
is  then  to  be  made  in  the  median  line  through  the  skin  and 
down  to  the  fasciae.  Two  subcutaneous  sutures,  as  em- 
ployed by  Halsted,  are  then  to  be  introduced  transversely 
to  the  line  of  incision  about  1  cm.  apart,  and  their  ends  left 


246 


BACTERIOLOGY 


loose.  This  particular  sort  of  suture  does  not  pass  through 
the  skin,  but,  instead,  the  needle  is  introduced  into  the 
subcutaneous  tissues  along  the  edge  of  the  incision.  In  this 
case  they  are  to  pass  into  the  abdominal  cavity  and  out 
again,  entering  at  one  side  of  the  line  of  incision  and  leaving 
at  the  other,  as  indicated  by  the  solid  and  dotted  lines  in 
Fig.  46.  (The  figure  indicates  the  primary  opening  through 
the  skin.  The  longitudinal  dotted  line  shows  the  opening 


FIG.  46 


Diagram  of  skin  incision  and  sutures  in  laparotomy  on  animals. 

to  be  made  into  the  abdomen;  the  transverse  dotted  lines, 
with  their  loose  ends,  represent  the  sutures  as  placed  in 
position  before  the  abdomen  is  opened;  it  will  be  seen  that 
these  sutures  in  all  cases  pass  through  the  subcutaneous 
tissues  only  and  do  not  penetrate  the  skin  proper.) 

The  opening  through  the  remaining  layers  may  now  be 
completed;  the  bit  of  tissue  is  deposited  in  the  peritoneal 
cavity,  under  precautions  that  will  exclude  all  else,  the 


SUBCUTANEOUS  INOCULATION  OF  ANIMALS     247 

edges  of  the  wound  drawn  evenly  and  gently  together  by 
tying  the  sutures,  and  the  lines  of  incision  dressed  with 
collodion.  It  should  be  needless  to  say  that  this  operation 
must  be  conducted  under  the  strictest  precautions,  to 
avoid  complications.  All  instruments,  sutures,  ligatures, 
etc.,  must  be  carefully  sterilized  either  in  the  steam  sterilizer 
for  twenty  minutes,  or  by  boiling  in  2  per  cent,  sodium 
carbonate  solution  for  ten  minutes;  the  hands  of  the  opera- 
tor, though  they  should  not  touch  the  wound,  must  be 
carefully  cleansed,  and  the  material  to  be  introduced  into 
the  abdomen  should  be  handled  with  only  sterilized  instru- 
ments. 

Inoculation  into  the  pleural  cavity  is  much  less  frequently 
required — in  fact,  it  is  not  a  routine  method.  It  is  not  easy 
to  enter  the  pleural  cavity  with  a  hypodermic  needle  without 
injuring  the  lung,  and  it  is  rare  that  conditions  call  for  the 
introduction  of  solid  particles  into  this  locality. 

Inoculation  into  the  anterior  chamber  of  the  eye  is  per- 
formed by  making  a  puncture  through  the  cornea  just  in 
front  of  its  junction  with  the  sclerotic,  the  knife  being 
passed  into  the  anterior  chamber  in  a  plane  parallel  to  the 
plane  of  the  iris.  By  the  aid  of  a  fine  pair  of  forceps  the  bit 
of  tissue  is  passed  through  the  opening  thus  made  and  is 
deposited  upon  the  iris,  where  it  is  allowed  to  remain,  and 
where  its  pathogenic  activities  upon  the  iris  can  be  con- 
veniently studied.  It  is  a  mode  of  inoculation  of  very 
limited  application,  and  is  therefore  but  rarely  practised. 
It  was  employed  in  the  classical  experiments  of  Cohnheim 
in  demonstrating  the  infectious  nature  of  tuberculous  tissues, 
tuberculosis  of  the  iris  being  the  constant  result  of  the 
introduction  of  tuberculous  tissue  into  the  anterior  chamber 
of  the  eye  of  rabbits. 


248  BACTERIOLOGY 

OBSERVATION  OF  ANIMALS  AFTER  INOCULATION. — After 
either  of  these  methods  of  inoculation,  particularly  when 
unknown  species  of  bacteria  are  being  tested,  the  animal  is  to 
be  kept  under  constant  observation  and  all  deviations  from 
the  normal  are  to  be  carefully  noted — as,  for  instance,  eleva- 
tion of  temperature;  loss  of  weight;  peculiar  position  in 
the  cage;  loss  of  appetite;  roughening  of  the  hair;  excessive 
secretions,  from  either  the  air-passages,  conjunctiva,  or 
kidneys;  looseness  of  or  hemorrhage  from  the  bowels; 
tumefaction  or  reaction  at  site  of  inoculation,  etc.  If  death 
ensue  in  from  two  to  four  days,  it  may  reasonably  be  expected 
that  at  autopsy  evidence  of  either  acute  septic  or  toxic 
processes  will  be  found.  It  sometimes  occurs,  however, 
that  inoculation  results  in  the  production  of  chronic  con- 
ditions, and  the  animal  must  be  kept  under  observation 
often  for  weeks.  In  these  cases  it  is  important  to  note  the 
progress  of  the  disease  by  its  effect  upon  the  physical  condi- 
tion of  the  animal,  viz.,  upon  the  nutritive  processes,  as 
evidenced  by  fluctuation  in  weight,  and  upon  the  body- 
temperature.  For  this  purpose  the  animal  is  to  be  weighed 
daily,  always  at  about  the  same  hour  and  always  about  mid- 
way between  the  hours  of  feeding;  at  the  same  time  its 
temperature,  as  indicated  by  a  thermometer  placed  in  the 
rectum,  is  to  be  recorded.1  By  comparison  of  these  daily 
observations  the  observer  is  aided  in  determining  the  course 
the  infection  is  taking. 

Too  much  stress  must  not,  however,  be  laid  upon  moderate 
and  sudden  daily  fluctuations  in  either  temperature  or 
weight,  as  it  is  a  common  observation  that  presumably 

1  The  thermometer  must  be  inserted  into  the  rectum  beyond  the  grasp 
of  the  sphincter,  otherwise  pressure  upon  its  bulb  by  contraction  of  this 
muscle  may  force  up  the  mercurial  column  to  a  point  higher  than  that 
resulting  from  the  actual  body-temperature. 


SUBCUTANEOUS  INOCULATION  OF  ANIMALS     249 

normal  animals  when  confined  in  cages  and  fed  regularly 
often  present  very  striking  temporary  gains  and  losses  in 
weight,  often  amounting  to  50  or  100  grams  in  twenty-four 
hours,  even  in  animals  whose  total  weight  may  not  exceed 
500  or  600  grams;  similarly  unexplainable  rises  and  falls 
of  temperature,  often  as  much  as  a  degree  from  one  day  to 
another,  are  seen.  Such  fluctuations  have  apparently  no 
bearing  upon  the  general  condition  of  the  animal,  but  are 
probably  due  to  transient  causes,  such  as  overfeeding  or 
scarcity  of  food,  improper  feeding,  lack  of  exercise,  excite- 
ment, fright,  etc. 

The  accompanying  charts  (Figs.  47,  48,  49,  50)  will  serve 
to  illustrate  some  of  these  points.  The  animals,  two  rabbits 
and  two  guinea-pigs,  were  taken  at  random  from  among 
stock  animals  and  placed  each  in  a  clean  cage,  the  kind  used 
for  animals  under  experiment,  and  kept  under  as  good 
general  conditions  as  possible.  For  the  first  week  the  rabbits 
received  each  100  grams  of  green  food  (cabbage  and  turnips) 
daily,  and  the  guinea-pigs  30  grams  each  of  the  same  food. 
During  the  second  week  this  daily  amount  of  food  was 
doubled;  during  the  third  week  it  was  quadrupled;  and  for 
the  fourth  and  fifth  weeks  they  each  received  an  excess  of 
food  daily,  consisting  of  green  vegetables  and  grains  (oats 
and  corn).  By  reference  to  the  charts  sudden  diurnal 
fluctuations  in  weight  will  be  observed  that  do  not  corre- 
spond in  all  instances  with  scarcity  or  sufficiency  of  food. 
With  the  rabbits  there  is  a  gradual  loss  of  weight  with  the 
smaller  amounts  of  food,  which  losses  are  not  totally  re- 
covered as  the  food  is  increased.  With  the  guinea-pigs  there 
is  likewise  at  first  a  loss;  but  after  a  short  time  the  weight 
remains  tolerably  constant,  and  is  not  so  conspicuously 
affected  by  the  increase  in  food  as  one  might  expect.  From 


250 


BACTERIOLOGY 


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SUBCUTANEOUS  INOCULATION  OF  ANIMALS     251 

presenting  such  fluctuations,  is  about  a  degree  or  more, 
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252 


BACTERIOLOGY 


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SUBCUTANEOUS  INOCULATION  OF  ANIMALS     253 

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254  BACTERIOLOGY 

susceptible,  rabbits  in  particular,  if  properly  fed,  will  fre- 
quently gain  steadily  in  weight.  The  condition  of  progressive 
emaciation  just  mentioned  is  conspicuously  seen  after 
intravenous  inoculation  of  rabbits  with  cultures  of  bacillus 
typhosus  and  of  bacillus  coli,  referred  to  in  the  chapter  on  the 
latter  organism,  and  if  looked  for  will  doubtless  be  seen  to 
follow  inoculation  with  other  organisms  capable  of  producing 
chronic  forms  of  infection,  but  which  are  frequently  con- 
sidered non-pathogenic  because  of  their  inability  to  induce 
acute  conditions.  Not  infrequently  in  chronic  infections 
there  may  be  hardly  any  marked  and  constant  temperature- 
variations  until  just  before  death,  when  sometimes  there 
will  be  a  rise  and  at  other  times  a  fall  of  temperature.  In 
the  majority  of  cases,  however,  one  must  be  very  cautious 
as  to  the  amount  of  stress  laid  upon  changes  in  weight  and 
temperature,  for  unless  they  are  progressive  or  continuous 
in  one  or  another  direction  they  may  have  little  significance 
as  indicating  the  existence  or  absence  of  disease. 


CHAPTER  XIII. 

Post-mortem  Examination  of  Animals — Bacteriological  Examination  of  the 
Tissues — Disposal  of  Tissues  and  Disinfection  of  Instruments  after 
the  Examination — Study  of  Tissues  and  Exudates  During  Life. 

DURING  bacteriological  examination  of  the  tissues  of  dead 
animals  certain  precautions  must  be  rigidly  observed  in 
order  to  arrive  at  correct  conclusions. 

The  autopsy  should  be  made  as  soon  as  possible  after 
death.  If  delay  cannot  be  avoided,  the  animal  should  be 
kept  on  ice  until  the  examination  can  be  made,  otherwise 
decomposition  sets  in,  and  the  saprophytic  bacteria  now 
present  may  interfere  with  the  accuracy  of  results.  When 
the  autopsy  is  to  be  made  the  animal  is  first  inspected 
externally,  and  all  visible  lesions  noted.  It  is  then  to  be 
fixed  upon  its  back  upon  a  board  with  nails  or  tacks.  The 
four  legs  and  the  end  of  the  nose,  through  which  the  tacks 
are  driven,  are  to  be  moderately  extended.  Plates  are  now 
to  be  made  from  the  site  of  inoculation,  if  this  is  subcuta- 
neous. The  surfaces  of  the  thorax  and  abdomen  are  then 
to  be  moistened  to  prevent  the  fine  hairs,  dust,  etc.,  from 
floating  about  in  the  air  and  interfering  with  the  work.  An 
incision  is  then  made  through  the  skin  from  the  chin  to  the 
symphysis  pubis.  This  is  only  a  skin  incision,  and  does  not 
reach  deeper  than  the  fascise.  It  is  best  done  by  first  making 
with  a  scalpel  an  incision  just  large  enough  to  permit  of  the 
introduction  of  one  blade  of  a  blunt-pointed  scissors.  It  is 
then  completed  with  the  scissors.  The  whole  of  the  skin  is 

(255) 


256  BACTERIOLOGY 

now  to  be  carefully  dissected  away,  not  only  from  the 
abdomen  and  thorax,  but  from  the  axillary,  inguinal,  and 
cervical  regions,  and  the  fore  and  hind  legs  as  well.  It  is 
then  pinned  flat  upon  the  board  so  as  to  keep  it  as  far  from 
the  abdomen  and  thorax  as  possible,  for  it  is  from  the  skin 
that  the  chances  of  contamination  are  greatest. 

It  now  becomes  necessary  to  proceed  very  carefully. 
All  incisions  from  this  time  on  are  to  be  made  only  through 
surfaces  that  have  been  sterilized.  The  sterilization  is  best 
accomplished  by  the  use  of  a  broad-bladed  table-knife  that 
has  been  heated  in  a  gas-flame.  The  blade,  made  quite  hot, 
is  to  be  held  upon  the  region  of  the  linea  alba  until  the  tissues 
of  that  region  begin  to  burn;  it  is  then  held  transversely 
to  this  line  over  about  the  center  of  the  abdomen,  thus 
making  two  sterilized  tracks,  through  which  the  abdomen 
may  be  opened  by  a  crucial  incision.  The  sterilization  thus 
accomplished  is,  of  course,  directed  only  against  organisms 
that  may  have  fallen  upon  the  surface  from  without,  and 
therefore,  it  need  not  extend  deep  down  through  the  tissues. 
In  the  same  way  two  burned  lines  may  be  made  from  either 
extremity  of  the  transverse  line  up  to  the  top  of  the  thorax. 

With  hot  scissors  the  central  longitudinal  incision  extend- 
ing from  the  point  of  the  sternum  to  the  genitalia  is  to  be 
made  without  touching  the  internal  viscera.  The  abdominal 
wall  must  therefore  be  held  up  during  the  operation  with 
sterilized  forceps  or  hooks.  The  cross-incision  is  made  in 
the  same  way.  When  this  is  completed  an  incision  through 
the  ribs  with  a  pair  of  heavy,  sterilized  scissors  is  made 
along  the  scorched  tracks  on  either  side  of  the  thorax.  After 
this  the  whole  anterior  wall  of  the  thorax  may  easily  be 
lifted  up,  and  by  severing  the  connections  with  the  dia- 
phragm it  may  be  completely  removed.  When  this  is  done 


POST-MORTEM  EXAMINATION  OF  ANIMALS     257 

and  the  abdominal  flaps  laid  back,  the  contents  of  both 
cavities  are  to  be  inspected  and  their  condition  noted  without 
disturbing  them. 

After  this  the  first  steps  to  be  taken  are  to  prepare  plates 
or  Esmarch  tubes  from  the  blood,  liver,  spleen,  kidneys, 
and  any  exudates  that  may  exist.  This  is  best  done  as 
follows:  Heat  a  scalpel  quite  hot  and  apply  it  to  a  small 
surface  of  the  organ  from  which  cultures  are  to  be  made. 
Hold  it  upon  the  organ  until  the  surface  directly  beneath 
is  visibly  scorched.  Then  remove  it,  heat  it  again,  and 
while  quite  hot  insert  its  point  through  the  capsule  of  the 
organ.  Into  the  opening  thus  made  insert  a  sterilized 
platinum  loop,  made  of  wire  a  little  heavier  than  that 

FIG.  51 


Nuttall's  platinum  spear  for  use  at  autopsies. 

commonly  employed.  Project  this  deeply  into  the  tissues 
of  the  organ;  by  twisting  it  about  enough  material  from 
the  center  of  the  organ  can  be  obtained  for  making  the 
cultures. 

As  the  resistance  offered  by  the  tissue  is  sometimes  too 
great  to  permit  of  puncture  with  the  ordinary  wire  loop, 
Nuttall1  devised  for  the  purpose  a  platinum-wire  spear 
which  possesses  great  advantages  over  the  loop.  It  has 
the  form  seen  in  Fig.  51.  It  is  easily  made  by  beating  a 
piece  of  heavy  platinum  wire  into  a  spear-head  at  one  end, 
and  perforating  this  with  a  small  drill,  as  seen  in  the  cut. 
It  is  attached  by  the  other  end  to  either  a  metal  or  glass 

1  Centralblatt  fur  Bakteriologie  und  Parasitenkunde,  1892,  Bd.  xi,  p.  538. 
17 


258  BACTERIOLOGY 

handle,  preferably  the  former.  It  can  readily  be  thrust  into 
the  densest  of  the  soft  tissues,  and  by  twisting  it  about 
after  its  introduction  particles  of  the  tissue  sufficient  for 
examination  are  withdrawn  in  the  eye  of  the  spear-head. 

Cultures  from  the  blood  are  usually  made  from  one  of  the 
cavities  of  the  heart,  which  is  always  punctured  at  a  point 
which  has  been  burned  in  the  way  given. 

In  addition  to  cultures,  cover-slips  from  the  site  of  inocu- 
lation, from  each  organ,  and  from  any  exudates  that  may 
be  present  must  be  made.  These,  however,  are  prepared 
after  the  materials  for  the  cultures  have  been  obtained. 
They  need  not  be  examined  immediately,  but  may  be 
placed  aside,  under  cover,  on  bits  of  paper  upon  which  the 
name  of  the  organ  from  which  they  were  prepared  is  written. 

When  the  autopsy  is  complete  and  the  gross  appearances 
have  been  carefully  noted,  small  portions  of  each  organ  are 
to  be  preserved  in  95  per  cent,  alcohol  for  subsequent 
examination.  Throughout  the  entire  autopsy  it  must  be 
borne  in  mind  that  all  cultures,  cover-slips,  and  tissues  must 
be  carefully  labelled,  not  only  with  the  name  of  the  organ 
from  which  they  originate,  but  with  the  date,  designation 
of  the  animal,  etc.,  so  that  an  account  of  their  condition 
after  closer  study  may  be  subsequently  inserted  in  the 
protocol. 

The  cover-slips  are  now  to  be  stained,  mounted,  and 
examined  microscopically,  and  the  results  carefully  noted. 

The  same  care  with  regard  to  noting,  labelling,  etc., 
should  be  exercised  in  the  subsequent  study  of  the  cultures 
and  the  hardened  tissues,  which  are  to  be  stained  and  sub- 
jected to  microscopic  examination.  The  results  of  micro- 
scopic study  of  the  cover-slip  preparations  and  of  those 
obtained  by  cultures  should  in  most  cases  correspond, 


POST-MORTEM  EXAMINATION  OF  ANIMALS      259 

though  it  not  rarely  occurs  that  bacteria  are  present  in 
such  small  numbers  in  the  tissues  that  their  presence  may 
be  overlooked  microscopically,  and  still  they  appear  in  the 
cultures. 

If  the  autopsy  has  been  performed  in  the  proper  way, 
with  the  precautions  given,  and  sufficiently  soon  after  death, 
the  results  of  the  bacteriological  examination  should  be 
either  negative  or  the  organisms  which  are  isolated  should 
be  in  pure  cultures.  This  is  particluarly  the  case  with  cul- 
tures made  from  the  internal  viscera. 

Both  the  cover-slips  and  cultures  made  from  the  point 
of  inoculation  are  apt  to  contain  a  variety  of  organisms. 

If  the  organism  obtained  in  pure  culture  from  the  internal 
viscera,  or  those  predominating  at  the  point  of  inoculation 
of  the  animal,  have  caused  its  death,  then  subsequent 
inoculation  of  pure  cultures  of  this  organism  into  the  tissues 
of  a  second  animal  should  produce  similar  results. 

When  the  autopsy  is  quite  finished  the  remains  of  the 
animal  should  be  burned;  all  instruments  subjected  to 
either  sterilization  by  steam  or  boiling  for  fifteen  minutes 
in  a  1  to  2  per  cent,  soda  solution;  and  the  board  upon  which 
the  animal  was  tacked,  as  well  as  the  tacks,  towels,  dishes, 
and  all  other  implements  used  at  the  autopsy,  be  sterilized 
by  steam.  All  cultures,  cover-slips,  and,  indeed,  all  articles 
likely  to  have  infectious  material  upon  them,  must  be 
sterilized  as  soon  as  they  are  of  no  further  service. 

What  has  been  said  with  regard  to  the  study  of  dead 
tissues  obtained  at  autopsy  applies  equally  well  to  the 
bacteriological  study  of  tissues  and  exudates  obtained  during 
life.  In  the  latter  case,  however,  certain  .precautions  are 
always  to  be  observed.  In  the  first  place,  it  is  desirable  to 


260  BACTERIOLOGY 

obtain  the  materials  under  aseptic  precautions,  care  being 
taken  that  no  disinfectant  fluids  are  mixed  with  them. 
They  should  be  subjected  to  study  as  soon  as  possible  after 
removal  from  the  body.  In  the  case  of  tissues  that  cannot 
be  examined  on  the  spot,  they  should  be  placed  in  a  sterile 
Petri  dish  or  in  a  stoppered,  sterile,  wide-mouthed  bottle 
and  taken  at  once  to  the  laboratory.  The  surface  should 
then  be  seared  with  a  hot  knife  and  an  incision  through  the 
seared  area  into  the  center  made  with  a  knife  that  has  been 
sterilized  and  allowed  to  cool.  From  the  depths  of  this 
incision  enough  material  may  be  obtained  for  microscopic 
examination  and  for  the  preparation  of  cultures.  Fluid 
exudates  that  must  be  taken  to  the  laboratory  should  be 
collected  in  either  a  sterile  test-tube,  or,  better,  in  a  sterile 
capillary  tube  that  is  subsequently  sealed  at  both  ends  in 
a  gas-flame.  When  bacteriological  examination  of  the 
blood  during  life  is  required,  it  is  customary  to  obtain  the 
necessary  sample  of  blood  by  pricking  the  skin.  It  must 
be  remembered,  in  this  connection,  that  the  skin  usually 
contains  a  number  of  species  of  bacteria  that  are  of  no 
pathological  significance  and  have  nothing  to  do  with  the 
disease  from  which  the  individual  may  be  suffering.  It  is 
manifestly  essential  to  exclude  these.  It  is  not  possible  to 
exclude  them  certainly  and  completely  under  all  circum- 
stances, without  a  more  or  less  elaborate  procedure;  but 
an  effort  to  do  so  should  always  be  made.  As  a  rule,  the 
greater  number  of  them  may  be  removed  from  the  skin  by 
careful  washing  with  warm  water  and  soap  and  a  sterile 
brush,  after  which  the  skin  should  be  rinsed  with  alcohol 
and  allowed  to  dry  spontaneously.  The  drop  of  blood  may 
then  be  obtained  from  the  skin  thus  cleaned  by  a  prick 
with  a  sharp,  sterilized  lancet.  The  presence  in  the  cultures 
of  a  staphylococcus,  growing  slowly,  with  white  colonies, 


ULTRA-MICROSCOPIC  OR  FILTERABLE   VIRUSES         261 

is  a  frequent  experience,  and  does  not  necessarily  imply 
that  this  organism  bears  an  etiological  relation  to  the  disease 
from  which  the  individual  may  be  suffering  (see  Staphylococcus 
Epidermis  Albus). 

When  more  than  a  few  drops  of  blood  are  needed,  as  may 
be  the  case  in  deciding  the  general  nature  of  an  infection 
process,  it  is  customary  to  withdraw  it  from  one  of  the  super- 
ficial veins  of  the  forearm  by  means  of  an  hypodermic 
syringe.  The  operation  should  be  done  under  strictly 
asceptic  conditions,  i.  e.,  the  skin  should  be  thoroughly 
cleaned  with  soap,  water,  and  alcohol;  the  hands  of  the 
operator  should  be  surgically  cleaii;  the  syringe  must  have 
been  sterilized  immediately  before  using,  and  great  care 
should  be  taken  that  no  air  bubbles  be  injected  into  the 
veins  during  the  operation. 

In  interpreting  the  results  of  cultures  made  from  blood 
drawn  in  this  manner,  the  possibility  of  contamination  by 
skin  bacteria  should  not  be  forgotten.  The  success  of  the 
operation  depends  upon  attention  to  the  most  minute 
details  of  aseptic  practice.  It  requires  for  its  safe  practice 
skill  in  manipulation,  experience  and  judgment  in  the  inter- 
pretation of  the  results.  It  is  not,  therefore,  an  operation 
to  be  commended  to  the  beginner. 

"ULTRA"-MICROSCOPIC   OR   "FILTERABLE"  VIRUSES. 

These  terms  relate  to  particular  substances  capable  of 
causing  disease,  that  are  so  small  as  to  be  beyond  the  visual 
range  of  the  microscopes  used  in  bacteriological  work,  which 
do  not  respond  to  the  usual  methods  for  the  cultivation  of 
bacteria  and  which  are  able,  because  of  their  minute  dimen- 
sions, to  pass  through  the  pores  of  the  finer  grades  of  earth- 
enware filters. 


262  BACTERIOLOGY 

Their  existence  has  been  suspected  for  a  number  of  years 
but  it  is  only  comparatively  recently  that  sufficient  became 
known  of  them  to  justify  our  speaking  confidently  of  them; 
and  even  now  little  more  than  their  etiological  potentialities 
and  some  of  their  physiological  reactions  can  be  considered. 

For  a  long  while  it  has  been  a  puzzle  that  such  character- 
istic contagious  diseases  as  certain  of  the  acute  exanthemata 
in  man  and  a  number  of  typical  transmissible  diseases  in 
animals  should  have  eluded  all  efforts  to  discover  their 
causes.  By  the  customary  methods  of  bacteriological 
analysis  nothing  of  a  positive  character  is  learned  and  yet 
by  the  introduction  into  susceptible  animals  of  bits  of  tissue 
from  the  diseased  animal,  or  small  quantities  of  blood  or 
tissue  juices  or  even  of  filtrates  of  emulsions  of  such  tissues 
or  juices,  it  is  possible  in  a  number  of  instances  to  reproduce 
the  disease.  It  is  such  evidence  as  this  that  serves  as  the 
basis  for  the  belief  in  the  existence  of  invisible  or  elusive 
viruses  for  a  number  of  diseases  of  man  and  animals  and 
a  few  for  plants. 

The  existence  of  such  viruses  has  been  demonstrated 
in  smallpox  vaccine,  measles,  typhus  fever,  dengue  fever, 
poliomyelitis,  and  trachoma,  among  the  diseases  of  man 
and  in  foot  and  mouth  disease,  contagious  pleuro-pneu- 
monia,  sheep-pox,  rabies,  cattle  plague,  chicken  sarcoma, 
and  distemper  of  dogs  among  those  of  animals,  and  in  the 
mosaic  disease  of  the  tobacco  plant.  Sometimes  such  fil- 
trates when  placed  under  special  methods  of  cultivation 
show  evidence  of  multiplication  by  clouding  of  the  media 
but  with  no  development  of  recognizable  morphological 
structures — in  a  few  instances  such  cultures  have  shown  the 
development  of  minute  spiral  forms  of  organisms.  (See 
Leptospira  icteroides. 

Though  little  or  nothing  that  is  convincing  can  be  said 


ULTRA-MICROSCOPIC  OR  FILTERABLE   VIRUSES      263 

of  the  morphology  of  this  group  of  ultra-microscopic  par- 
ticles, still  in  their  reactions  to  a  variety  of  physical  agents 
they  are  obviously  living  matter,  having  many  analogies  to 
the  more  highly  developed  microorganisms  with  which  we 
are  familiar.  Practically  all  are  killed  at  temperatures 
ranging  from  55°  to  70°  C.  Some  resist  drying  for  com- 
paratively long  periods  of  time,  others  are  quickly  killed  by 
it.  Practically  all  are  resistant  to  the  action  of  glycerin. 
This  is  not  the  case  as  a  rule  with  bacteria.  They  vary 
considerably  in  their  resistance  to  such  germicidal  substances 
as  formalin,  boric  acid,  corrosive  sublimate  and  menthol. 

Practically  all  animals  that  survive  their  invasion  have 
acquired  immunity  from  a  second  attack  of  the  disease. 
There  is  little  evidence  that  the  growth  is  accompanied  by 
the  production  of  toxins  as  such.  A  survey  of  such  data  as 
are  available  justifies  the  suspicion  that  these  bodies  are 
more  closely  allied  to  the  protozoa  than  to  the  bacteria. 

Efforts  at  cultivation  under  artificial  circumstances  have 
succeeded  in  only  a  few  instances.  In  their  studies  upon  the 
contagious  pleuro-pneumonia  of  cattle  Nocard  and  Roux 
by  the  use  of  special  methods,  both  optical  and  cultural, 
claim  to  have  demonstrated  the  causative  factor  of  that 
disease.  The  method  employed  by  them  for  the  cultivation 
of  the  virus  is  that  suggested  by  Metchnikoff,  Roux  and 
Salambini  in  1896.  It  consists  in  placing  bits  of  tissue  or 
secretions  from  the  infected  animals  in  small,  sterilized 
collodion  sacs,  which  are  finally  hermetically  sealed  with 
sterile  collodion.  These  little  sacs  with  their  contents  are 
then  placed  in  the  peritoneal  cavity  of  an  animal;  a  rabbit, 
chicken,  guinea-pig,  calf,  dog,  or  sheep  as  the  case  may  be, 
and  left  there  for  a  time.  The  idea  on  which  this  method  is 
based  is  that  the  collodion  sacs  are  impermeable  for  the 
specific  virus  but  are  permeable  to  the  normal  juices  of  the 


264  BACTERIOLOGY 

peritoneal  cavity  of  the  animal  in  which  they  are  placed. 
Under  these  circumstances  the  specific  virus  was  expected  to 
develop  within  the  sacs  and  receive  its  food  supply  by  dif- 
fusion from  the  surrounding  peritoneum;  the  body  tem- 
perature of  the  animal  in  which  they  were  placed  being  most 
favorable  to  incubation. 

The  investigators  found  that  by  the  use  of  a  special 
system  of  illumination  and  very  high  magnification,  about 
2000  diameters,  there  were  to  be  detected  within  the  col- 
lodion sacs,  in  from  a  few  days  to  several  weeks,  numerous 
motile  points  or  dots  of  such  minute  dimensions  that  it  was 
often  impossible  to  decide  as  to  their  exact  form.  No  such 
bodies  were  seen  in  control  collodion  sacs  placed  similarly 
in  the  peritoneum  of  animals  but  in  which  sacs  none  of  the 
tissue  or  juices  from  a  diseased  animal  had  been  inclosed. 
Nocard  and  Roux  are  disposed  to  regard  these  bodies  as 
the  exciting  cause  of  the  disease  under  consideration. 

Flexner  and  Noguchi  announce  that  by  the  use  of  Nogu- 
chi's  method  for  cultivating  spirochetse  (see  Spirochetacese) 
they  have  isolated  from  the  central  nervous  tissues  of  both 
man  and  monkeys  dead  of  poliomyelitis,  minute  coccus-like 
bodies  that  they  believe  to  be  the  cause  of  the  disease. 
The  culture  medium  consists  of  human  ascetic  fluid  to  which 
a  fragment  of  sterile  fresh  rabbit  kidney  has  been  added. 
The  cultivation  is  conducted  at  first  under  anaerobic  condi- 
tions but  later  subcultures  do  not  demand  complete  absence 
of  free  oxygen.  When  ready  the  tubes  are  inoculated  with 
small  bits  of  the  diseased  cerebrum  or  cord  after  which  a 
thick  layer  of  sterile  paraffin  oil  is  placed  upon  the  surface 
of  the  ascetic  fluid.  This  suffices  for  the  exclusion  of  free 
oxygen. 

After  from  seven  to  twelve  days  at  body  temperature  a  dif- 
fuse clouding  or  opalescence  appears  about  the  bit  of  nervous 


ULTRA-MICROSCOPIC  OR  FILTERABLE  VIRUSES     265 

tissue  in  the  tube.  Microscopic  examination  of  this  opalescent 
matter,  especially  by  dark-field  illumination,  reveals  the  pres- 
ence of  coccoid  bodies  conspicuous  for  their  variation  in  size. 

Their  true  nature  has  not  been  determined.  The  disease 
can  be  reproduced  in  monkeys  by  inoculation  with  the  cul- 
tures, but  not  with  regularity.1 

By  an  analogous  method  Noguchi  has  cultivated  from 
both  rabies  and  trachoma  bodies  that  he  regards  as  etiolog- 
ically  related  to  the  diseases  from  which  they  were  obtained. 
It  is  not  possible  as  yet  to  be  either  certain  as  to  the  accuracy 
of  his  suspicions  or  to  satisfactorily  classify  the  bodies  found 
in  his  cultures.  In  some  respects  they  suggest  bacteria,  in 
some  protozoa  and  taking  them  in  conjunction  with  the 
tissue  findings  in  the  diseases  it  seems  fair  to  suspect  that 
they  may  be  developmental  forms  of  the  Negri  bodies  con- 
stantly present  in  rabies  in  the  one  case  or  the  singular  cell 
inclusions  common  to  trachoma,  the  so-called  "trachoma 
bodies"  in  the  other.2 

In  the  study  of  many  of  the  common  diseases,  notably 
the  exanthemata,  both  at  autopsy  and  during  life,  by  the 
methods  above  outlined,  the  investigation  often  yields 
negative  results,  and  yet  there  is  every  reason  for  believing 
these  diseases  to  be  dependent  for  their  existence  upon 
invasion  of  the  body  by  some  form  or  another  of  living 
microorganisms,  capable  of  growth  in  the  tissues  and  sus- 
ceptible of  being  transmitted  from  individual  to  individual, 
either  directly  or  indirectly.  It  is  possible  that  the  applica- 
tion of  one  or  another  of  the  foregoing  methods  to  the 
study  of  these  diseases  may  demonstrate  that  some  of  them 
at  least  are  due  to  the  presence  of  so-called  filterable  viruses. 

1  For  details  see  Flexner  and  Noguchi,  Jour.  Exp.  Med.,  1913,  vol.  xviii, 
No.  4. 

2  For  particulars  see  Noguchi,  Jour.  Exp.  Med.,  1913,  No.  4;   ibid.,  1913, 
No.  5. 


CHAPTER  XIV. 

Infection  and   Immunity — Mechanism — Specific   Bodies    and   Reactions — 
Doctrines  in  Explanation. 

INFECTION.      . 

IF  one  examine  in  detail  the  lesions  resulting  from  the 
invasion  of  the  body  by  the  different  types  of  infective 
bacteria,  justification  is  found  for  -the  conclusion  that  the 
physical  manifestations  of  infection,  that  is,  the  sites  of 
activity  and  the  characteristic  lesions,  vary  with  the  nature 
of  the  different  invading  parasites. 

To  a  certain  extent  this  is  true;  that  is  to  say,  the  type 
of  lesion  characterizing  a  specific  disease  is  peculiar  to  that 
disease  and  is  produced  only  by  the  particular  microorganism 
having  the  power  to  excite  the  disease.  But  if  we  take  up 
the  various  lesions  of  specific  diseases  in  intimate  detail  we 
shall  see,  as  will  be  shown  later,  that  fundamentally  the 
essential  factor  in  the  mechanism  of  infection  is  of  the  same 
general  nature  for  all*  diseases,  be  the  characteristic  lesions 
and  clinical  manifestations  what  they  may;  the  apparent 
differences  being  referable  to  dissimilarities  of  structure  and 
function  of  the  various  species  of  bacteria  that  excite  the 
several  phenomena  on  the  one  hand,  and  to  the  parts  of 
the  body  of  the  host  that  are  attacked  on  the  other.  Thus, 
by  way  of  illustration,  if  we  select  a  group  of  clinically  and 
pathologically  distinct  infections,  such  as  anthrax,  miliary 
tuberculosis,  and  diphtheria,  and  compare  the  conditions 

recorded  at  autopsy,  little  of  a  macroscopic  nature  will  be 
(266) 


INFECTION  267 

discovered  to  suggest  anything  that  is  common  to  all,  and 
even  if  the  tissues  be  examined  microscopically  such  marked  • 
divergencies  are  seen  that  we  are  still  in  doubt  as  to  the 
existence  of  a  common  factor.  In  the  case  of  anthrax,  a 
true  septicemia,  the  blood  current  is  the  seat  of  activity  of 
the  exciting  bacteria,  and  beyond  congestion,  enormous 
numbers  of  bacteria  in  the  bloodvessels  and  the  escape  of 
serum  into  the  tissues  (edema),  little  else  is  to  be  seen  to 
account  for  death.  On  the  other  hand,  in  the  case  of  miliary 
tuberculosis,  even  though  the  involvement  of  the  organs  may 
be  general,  there  is  no  similar  invasion  of  the  blood  stream. 
The  tubercles  are  circumscribed,  are  often  surrounded  by 
healthy  tissue  and,  though  obviously  distributed  throughout 
the  body  from  a  primary  focus  through  the  agency  of  the 
circulating  fluids,  each  tubercle  may  nevertheless  be  regarded 
as  a  distinct  local  infection.  There  is,  however,  a  conspicuous 
difference  between  the  lesions  found  here  and  those  seen  in 
anthrax.  The  lesion  of  tuberculosis,  the  tubercle,  is  always 
characterized  by  tissue  death  at  and  about  its  center,  i.  e., 
where  the  bacilli  are  located,  even  in  the  .earliest  stages  of 
its  development. 

On  postmortem  examination  of  an  animal  dead  of  diph- 
theria we  observe  conditions  that  are  unlike  those  noted  in 
both  anthrax  and  tuberculosis.  There  is  neither  an  invasion 
of  the  vascular  system  nor  a  distribution  of  conspicuous 
pathological  foci  throughout  the  body.  The  bacteria  are 
confined  to  the  primary  site  of  invasion  and  when  found  in 
distal  organs  are  there  only  in  small  numbers  and  give  no 
evidence  of  an  effect  upon  the  tissues  immediately  surround- 
ing them. 

Thus  far,  as  a  result  of  this  review,  we  have  two  points 
in  common  to  the  three  distinct  diseases,  viz.:  they  are  all 


268  BACTERIOLOGY 

caused  by  bacteria,  and  they  all  may  terminate  fatally. 
On  the  other  hand  the  clinical  symptoms  and  the  pathological 
lesions  are  such  as  to  characterize  each  as  a  pathological 
entity.  But,  as  has  been  intimated,  there  is  a  fundamental 
factor  common  to  all,  and  the  discovery  of  this  factor  gives 
the  clue  to  the  true  mechanism  of  all  infections.  Light  upon 
this  phase  of  the  subject  can  best  be  secured  through  experi- 
mental methods. 

Observation  and  experiment  have  taught  us  that  some- 
times highly  pathogenic  bacteria  may  lose  in  part  or  in 
whole  their  disease  producing  properties  without  at  the  same 
time  losing  their  vitality.  If  such  "attenuated"  bacteria 
be  injected  into  susceptible  animals  the  result  may  be 
nothing;  or  it  may  be  a  modified  lesion  totally  dissimilar 
to  that  following  injection  of  the  fully  virulent  organism. 
This  is  often  the  case  with  the  bacteria  that  excite  septicemia, 
and  the  bacillus  causing  anthrax  serves  as  a  useful  illustra- 
tion. When  normal,  as  it  is  usual  to  regard  it,  it  is  fully 
virulent  and  causes  fatal  blood  poisoning  in  suceptible 
animals,  but  if  subjected  to  certain  chemical  or  physical 
influences  the  virulence  may  gradually  be  lessened  until 
finally  we  may  have  a  living  anthrax  bacillus  that  has  been 
deprived  of  almost  all  its  disease  producing  power.  If 
animals  be  inoculated  with  such  attenuated  anthrax  bacilli 
the  conditions  found  may  be  in  striking  contrast  to  those 
produced  by  the  normal  germ.  Instead  of  the  bloodvessels 
being  almost  packed  with  bacteria,  they  may  contain  few 
or  none,  and  the  only  bacteria  to  be  found  in  the  body  in 
numbers  are  at  and  immediately  about  their  point  of  deposit. 
Yet  these  animals  exhibit  clinical  symptoms  and  occasionally 
die. 

Similarly,  in  other  varieties  of  septicemia,  the  so-called 


INFECTION  269 

"  hemorrhagic  group"  we  see  as  a  rule  typical,  fatal  septice- 
mias  resulting  from  the  invasion  of  the  body  by  the  organisms 
causing  them;  but  at  times,  through  influences  not  fully 
known,  these  organisms  become  modified  in  their  physio- 
logical functions  so  that  instead  of  the  customary  general 
invasion  of  the  circulating  fluids  there  may  be  only  a  very 
slight  invasion  and  the  results  of  their  inoculation  are  prin- 
cipally-evidenced as  local  destruction  of  tissue,  sometimes 
with  fatal  results.  Obviously  then  these  organisms  have  the 
power  of  causing  constitutional  disturbances,  tissue  changes 
and  even  fatal  results  without  the  necessity  of  their  being 
themselves  disseminated  throughout  the  body  by  way  of 
the  circulating  fluids. 

As  said  above  the  characteristic  lesion  of  tuberculosis  is 
the  tubercle,  and  the  peculiarity  of  the  tubercle  is  necrosis, 
observable  almost  from  the  moment  it  begins  to  develop. 
If  tuberculosis  be  induced  through  the  intravenous  injection 
of  rabbits  with  carefully  prepared  suspensions  of  living  viru- 
lent tubercle  bacilli  the  resulting  miliary  tubercles  are  always 
marked  by  more  or  less  death  of  tissue  at  and  about  their 
center,  which  tissue  death  progresses  as  the  disease  progresses, 
until  it  reaches  a  point  easily  seen  with  the  naked  eye  and 
finally  incompatible  with  life.  If  on  the  other  hand  a  similar 
injection  be  made  with  a  suspension  of  tubercle  bacilli  that 
have  been  killed,  by  heat  or  otherwise,  disseminated  nodules, 
tubercles,  will  also  be  found  in  the  internal  organs.  These 
may  be,  histologically,  strikingly  like  those  following  the  use 
of  the  living  organism;  they  are  marked  by  the  characteristic 
tissue  death,  but  it  is  less  in  evidence  and  it  is  not  progres- 
sive beyond  certain  limits  and  the  injection  does  not  neces- 
sarily prove  fatal  to  the  animal.  As  a  result  of  this  experi- 
ment we  see  that  dead  bacteria  may  produce  a  result  differing 


270  BACTERIOLOGY 

only  in  degree  from  that  caused  by  the  same  species  when 
living  and  fully  virulent. 

A  similar  property  may  be  demonstrated  in  a  number  of 
other  pathogenic  species  in  no  way  related  to  bacillus  tuber- 
culosis. Obviously,  there  is  something  within  or  associated 
with  these  bacteria  that  may  act  upon  the  tissues  even 
though  the  bacteria  themselves  may  be  dead. 

In  our  autopsy  on  the  animal  dead  of  diphtheria  we  saw 
that  the  bacilli  were  not  distributed  throughout  the  body,  but 
were  confined  to  the  site  of  inoculation.  We  saw  at  the 
site  of  inoculation  a  tissue  reaction  scarcely  sufficient  to 
account  for  the  fatal  result,  yet  that  result  occurred  within 
a  comparatively  short  time  after  inoculation. 

When  diphtheria  occurs  in  human  beings  the  same  holds 
true  as  a  rule,  and  while  occasionally  the  local  reaction  in 
the  throat  is  such  as  gravely  to  imperil  life  through  obstruc- 
tion to  respiration,  the  real  danger  in  most  cases  is  not  local 
but  remote,  and  the  clinical  observations  on  the  living  subject 
affected  with  this  disease  point  to  the  far-reaching  influence 
of  a  local  phenomenon,  that,  of  itself,  may  often  seem  to  be 
of  but  slight  significance. 

If  the  internal  organs  of  either  animals  or  human  beings 
that  have  died  of  diphtheria  be  examined  microscopically, 
changes  are  easily  to  be  discovered  that  are  incompatible 
with  life  and  that  at  once  account  for  many  of  the  clinical 
manifestations  of  the  disease,  yet  these  changes  are  not 
accompanied  by  the  presence  of  bacteria  nor  by  any  other 
agent  that  can  be  detected  by  the  eye. 

It  is  plain,  then,  that  the  serious  influence  of  the  local 
infection  of  diphtheria  is  referable  to  a  something  that 
originates  at  the  point  where  the  bacteria  are  growing  and  is 
from  that  point  distributed  to  the  distant  organs. 


INFECTION  271 

Has  the  specific  germ  of  diphtheria  any  property  to  war- 
rant such  a  view?  If  a  fluid  culture  of  bacillus  diphtherise 
be  filtered  through  a  porcelain  filter,  the  filtrate  will  contain 
none  of  the  bacteria.  If  this  filtrate,  free  of  all  bacteria, 
be  injected  into  animals,  death  ensues;  and  if  the  tissues  of 
these  animals  be  examined,  all  of  the  most  important  lesions 
that  characterized  the  tissues  of  the  animal  dead  after 
inoculation  with  the  living  germ  are  to  be  found. 

If  a  parallel  experiment  be  made  with  the  bacillus  of 
tetanus  analogous  results  will  be  obtained. 

It  is  clear,  then,  that  here  are  two  species  of  bacteria  that 
excite  the  characteristic  results  through  the  instrumentality 
of  a  something  that  they  manufacture  in  the  course  of  their 
growth;  that  may  be  separated  from  them  by  the  simple 
process  of  filtration,  and  that  when  so  separated  possesses 
all  the  properties  of  specific  intoxicants. 

In  anthrax  and  other  septicemias  we  saw  that,  normally, 
the  infection  was  characterized  by  the  distribution  of  the 
bacteria  throughout  the  body,  but  that  modified  results, 
differing  only  in  degree,  might  still  be  obtained  with  the 
attenuated  organisms  without  such  general  distribution. 
These  latter  conditions  must,  therefore,  have  been  caused 
by  a  poison  elaborated  by  or  escaping  from  the  locally 
deposited  organisms  and  carried  to  distant  parts  of  the  body 
by  the  circulating  fluids.  In  tuberculosis  the  nodules  result- 
ing from  inoculation  with  the  dead  bacteria  must  have  been 
the  result  of  a  poison  associated  with  the  bodies  of  those 
dead  bacteria  and  liberated  with  their  disintegration  in  the 
tissues;  while  in  diphtheria  it  is  plain  that  its  characteristic 
manifestations  are  the  outcome  of  a  poison  produced  locally 
by  the  growing  bacteria  and  carried  thence  by  the  circulating 
fluids  to  distant  organs,  there  to  exhibit  its  destructive 
properties. 


272  BACTERIOLOGY 

Thus  far,  then,  infection  must  be  viewed  as  a  conflict 
between  bacteria  on  the  one  hand  and  tissues  on  the  other; 
the  former  having  as  their  weapons  of  offence  destructive 
poisons;  the  latter,  vital  defensive  provisions  that  enable 
them  to  resist  infection  with  greater  or  less  degree  of  success, 
according  to  circumstances.  It  makes  no  difference,  there- 
fore, whether,  in  infection,  the  bacteria  be  generally  or  only 
locally  present,  the  mechanism  of  infection  is  at  bottom  a 
destructive  intoxication. 

Bacterial  Toxins. — The  term  "toxins,"  as  used  in  bac- 
teriology, refers  to  a  group  of  soluable,  nitrogenous,  non- 
crystallizable  poisons  that  are  elaborated  by  certain  bacteria 
in  the  course  of  their  growth,  both  in  the  tissues  of  the  living 
host  and  under  conditions  of  artificial  cultivation.  They 
are  assumed  to  be  by-products  of  metabolism  and  they  may 
be  separated  easily  from  the  living  bacteria  by  which  they 
are  manufactured  by  the  simple  process  of  filtration,  through 
fine-pore  earthenware  filters.  As  they  have  not  been  ob- 
tained in  a  pure  state  their  chemical  composition  cannot  be 
stated  precisely  but  it  is  probable  that  they  are  allied  to  the 
globulins,  nucleo-albumens,  peptones,  albumoses,  or  the 
enzymes. 

The  toxins  are  identified,  not  by  their  chemical  structure, 
but  rather  by  their  harmful  action  upon  the  tissues  of  living 
animals,  i.  e.,  by  their  physiological  reactions.  It  is  this 
property  that  renders  them  of  such  significance  in  the 
phenomena  designated  as  disease. 

By  the  injection  of  either  of  these  bacteria-free,  true 
toxins  into  the  tissues  of  susceptible  animals,  lesions  are 
produced  that  are  in  all  essential  respects  identical  with 
those  occurring  in  the  course  of  infection  by  the  living  bac- 
teria. By  varying  the  dose  of  toxin  injected  into  the  animal 


INFECTION  273 

one  may  produce  either  prompt  death  or  only  slight  con- 
stitutional reaction.  In  the  latter  event  repeating  the 
injection  of  a  non-fatal  dose  may  have  no  apparent  effect 
upon  the  animal.  In  such  a  case  the  animal  has  acquired, 
loosely  speaking,  a  tolerance  to  the  poison  and  this  tolerance 
is  due  to  a  newly  formed,  antidotal  substance  now  circulating 
in  the  blood  of  the  tolerant  or  immune  animal.  For  example : 
If  a  measured  quantity  of  the  toxin  under  consideration  be 
mixed  in  test-tubes  with  varying  amounts  of  the  serum  of 
the  tolerant  animal  and  each  of  these  mixtures  be  injected 
into  fresh,  normal  animals  of  the  same  species,  it  will  be 
seen  that  in  some  instances  the  toxicity  of  the  poison  is 
only  lessened,  while  in  others  it  may  be  completely  neutral- 
ized; in  other  words,  we  have  demonstrated  by  such  an 
experiment  the  presence  in  the  blood  of  an  antidote,  and 
"antitoxin''  as  it  is  called.  This  antidote  is  specific,  that 
is,  it  can  neutralize  only  the  poison  used  in  the  experiment; 
it  is  inactive  when  used  against  other  toxins. 

This  union  between  toxin  and  its  antidote  is  conceived  to 
occur  according  to  the  laws  governing  ordinary  chemical 
reactions,  i.  e.,  there  is  a  definite  numerical  relationship; 
a  certain  fixed  quantity  of  toxin  being  neutralized  by  a 
certain  fixed  amount  of  antitoxin,  variations  in  either  factor 
resulting  in  failure  to  accurately  neutralize.  The  union 
between  the  two  factors  is  made  possible,  according  to 
Ehrlich's  conception,  through  the  possession  by  the  toxin 
molecule  and  by  the  antitoxin  molecule  of  constituents 
having  the  combining  function,  "  haptophore"  side  chains, 
as  he  calls  them.  In  addition  the  toxin  molecule  possesses 
another  constitutent  having  the  poisonous  destructive  func- 
tion, the  "toxiphoric,"  side  chains,  while  the  antidotal  or 
antitoxic  molecule  possesses  a  constitutent  having  the  neu- 
18 


274  BACTERIOLOGY 

tralizing  function.  Of  the  functions  of  these  side  chains, 
that  of  combination  is  the  more  permanent. 

Toxoids  and  Toxones. — Bearing  this  matter  of  permanency 
in  mind  we  find  that  when  toxins  are  allowed  to  stand, 
acted  upon  by  heat,  light  and  air,  for  a  time,  they  may 
still  combine,  as  may  be  determined  numerically,  with  the 
appropriate  antidotes  or  antitoxins,  but  may  show  evidence 
of  diminution  of  their  intoxicating  principle.  When  in  this 
degenerated  state  they  are  designated  as  "toxoids"  and 
"toxones." 

A  point  of  peculiar  interest  in  connection  with  the  true 
bacterial  toxins  is  the  extraordinary  toxicity  of  thqse  with 
which  we  are  more  or  less  fully  acquainted.  Experiment 
leads  to  the  belief  that  the  toxins  of  diphtheria  and  of  tetanus 
are  more  highly  poisonous  than  any  other  known  poisons. 
Thus,  for  instance,  diphtheria  toxin  is  capable  of  causing 
fatal  intoxication  in  a  guinea-pig  weighing  400  grams  when 
injected  subcutaneously  in  so  small  a  dose  as  0.05  milli- 
gram,1 while  typical  tetanus  is  produced  in  a  mouse  by  the 
injection  of  0.0001  milligram  of  tetanus  toxin.2 

The  number  of  bacteria  capable  of  elaborating  true  toxins 
is  very  small;  indeed,  in  so  far  as  those  of  significance  to 
animal  pathology  is  concerned,  we  are  certain  of  only  two 
species  having  this  property,  viz.,  the  bacillus  of  diphtheria 
and  the  bacillus  of  tetanus.  For  most  of  the  other  pathogenic 
species  their  toxic  action  is  referable,  not  to  toxins,  as  defined 
above,  but  rather  to  toxic  components  of  the  bacterial  cells, 
the  endotoxins  or  intracellular  toxins. 

The  Endotoxins  or  Intracellular  Toxins. — The  term  Endo- 
toxin  is  generically  used  to  designate  a  toxic,  protein  com- 
ponent of  the  bacterial  cells,  i.  e.,  it  is  part  and  parcel  of  the 

1  Roux  and  Yersin,  Annals  de  1'Inst.  Pasteur,  1889,  iii,  p.  287. 

2  Brieger  and  Cohn,  Zeit.  f.  Hyg.  u.  Infekt.,  1893,  Bd.  xv,  Heft  1, 


INFECTION  275 

cell  and  becomes  active,  presumably,  only  when  the  cells 
are  disintegrated.  Such  disintegration  may  occur  as  a 
result  of  autolysis  or  self-digestion  of  the  bacteria  under 
special  conditions  of  artificial  cultivation,  or  it  may  be  seen 
as  the  outcome  of  the  lytic  or  solvent  action  of  the  resisting 
body  cells  or  fluids,  either  those  of  the  infected  animal  or, 
as  in  the  case  of  the  toxins  and  antitoxins,  those  of  the 
animal  that  has  become  tolerant  in  one  way  or  another  to 
the  activities  of  the  bacteria  in  question.  Endotoxins  are 
not  liberated  from  the  bacterial  cells  as  a  secretion  or  excre- 
tion or  manufactured  as  an  extracellular  by-product,  as  is 
the  case  of  the  toxins,  but  are  peculiarities  of  the  protoplasm 
of  which  the  bacteria  are  composed. 

The  escape  of  endotoxin  from  bacterial  cells  as  a  result 
of  autolysis  is  seen  occasionally  in  old  cultures  that  have  been 
kept  for  a  time  under  more  or  less  constant  conditions. 
It  is  probable  that  it  occurs  to  a  limited  degree  in  all  cul- 
tures of  endotoxic  bacteria  as  a  result  of  the  death  and  final 
dissolution  of  a  smaller  or  larger  number  of  individual 
bacteria  in  such  cultures.  For  want  of  a  better  interpre- 
tation this  liberation  is  supposed  to  be  the  result  of  a  sort 
of  self-digestion  by  enzymes  that  are  within  the  bacteria 
as  normal  components.  It  is  most  conspicuously  to  be  seen 
in  cultures  of  those  endotoxic  species  that  most  readily  under- 
go those  morphological  changes  commonly  denominated  as 
involution  or  degeneration;  the  spirillum  of  Asiatic  cholera 
and  the  meningococcus  may  be  cited  as  conspicuous  illus- 
trations. The  fundamental  mechanisms  of  this  phenomenon 
cannot  be  discussed  with  profit  as  little  or  nothing  is  known 
of  it. 

As  in  the  case  of  toxins,  the  definite  chemical  nature  of 
endotoxins  cannot  be  stated.  Nevertheless  Buchner  isolated 


276  BACTERIOLOGY 

from  a  number  of  bacterial  species  protein  constituents, 
"bacterio-proteins,"  as  he  denominated  them,  having  the 
common  properties  of  soluability  in  alkalies,  relative  resist- 
ance to  the  boiling  temperature,  attraction  for  leukocytes 
(positive  chemotaxis),  and  pyogenic  powers. 

The  liberation  of  endotoxins  from  the  bacterial  cells  by 
strictly  bacteriolytic  processes  going  on  in  the  living  body 
is  not  a  simple  phenomenon.  It  is  conceived  as  resulting 
from  a  solution  of  the  bacteria  by  a  certain  ferment-like 
body  in  the  blood.  This  ferment-like  body  can  act  only 
when  it  is  bound  to  the  bacterial  cell  by  a  4  specific  inter- 
mediary body.  This  latter  is  supposed  to  be  portions  of 
cells  that  have  been  thrown  off  from  fixed  cells  in  the  course 
of  immunization,  i.  e.,  in  the  course  of  acquiring  tolerance 
to  the  action  of  certain  endotoxic  bacteria. 

In  numerous  instances  bacteria  are  disintegrated  by 
normal  blood.  Here  it  is  believed  that  the  ferment-like  body 
is  brought  into  action  through  the  agency  of  intermediary 
bodies  of  a  non-specific  nature,  i.  e.,  of  bodies  normally 
present  that  may  have  the  power  to  bind  any  or  all  bacteria 
to  the  ferment-like  body  and  thus  lead  to  their  destruction. 

While  the  blood  of  all  animals  possesses  some  destructive 
solvent  or  disintegrating  action  for  most  bacteria,  this  is 
never  so  great  as  is  that  of  the  blood  of  immunized  animals 
upon  the  particular  bacteria  from  which  they  are  immune. 

Endotoxins  Distinct  in  their  Action  from  Toxins. — Like 
toxins,  endotoxins,  i.  e.,  dead  endotoxic  bacteria,  may  cause 
disease  and  death  of  the  animal  tissues.  Similarly,  when 
endotoxic  bacteria  are  repeatedly  injected  in  sublethal 
doses,  immunity  of  varying  degrees  develops.  The  immunity 
resulting  from  the  use  of  non-fatal  doses  of  endotoxic  bac- 
teria, is  not,  however,  an  antitoxic  or  an  anti-endotoxic 


THE  DEFENSES  OF  THE  BODY  277 

immunity,  but  the  substance  appearing  in  the  blood  of 
animals  so  immunized  is  rather  bacteriolytic,  and  the  blood 
of  such  animals  may  contain  little  or  no  true  antitoxic 
components. 

Moreover,  if  the  blood  serum  of  an  animal  immune  from 
true  toxin  be  injected  into  a  normal  animal,  this  latter  at 
once  acquires  some  degree  of  resistance  to  the  toxin  from 
which  the  first  animal  was  protected,  i.  e.,  it  is  "passively" 
immunized;  on  the  other  hand,  if  the  bacteriolytic  serum  of 
an  animal  artificially  immunized  from  endotoxic  bacteria 
be  similarly  transferred  to  a  normal  animal,  there  is  no 
certainty  of  a  transference  of  the  state  of  immunity;  there 
may  be  a  transference  of  the  reaction  or  of  the  reacting  factors 
but  not  necessarily  of  a  protective  influence. 

THE   DEFENSES   OF   THE   BODY. 

When  considered  in  the  most  comprehensive  way  we  find 
that  the  normal  body  is  endowed  with  a  number  of  natural 
provisions  that  may  fairly  be  regarded  as  defenses  against 
the  invasion  of  hurtful  parasites.  Thus  for  instance:  If 
the  skin  of  even  the  most  cleanly  persons  be  examined 
bacteriologically,  we  find  that  in  the  majority  of  cases  bac- 
teria of  several  kinds,  often  those  having  the  power  to  cause 
disease,  are  to  be  detected.  So  long  as  the  skin  is  intact 
and  the  individual  in  good  general  health  no  harm  results. 
The  reason  for  this  is  found  in  the  structure  of  the  skin. 
The  horny  epidermis  and  the  fat  and  sweat  secretions  serve 
as  effectual  barriers  against  both  the  multiplication  of  germs 
and  their  penetration  into  the  underlying  tissues.  The  hairs 
about  the  orifices  act  to  some  extent  as  filters  or  screens  for 
bacteria  laden  dust;  the  ciliated  epithelium  of  the  upper  air 


278  BACTERIOLOGY 

passages  serves  as  a  sweep  to  rid  the  body  of  foreign  par- 
ticles that  may  find  lodgment  upon  it;  and  the  acid  reaction 
of  the  gastric  juice,  low  though  it  be,  is  thought  sufficient 
to  render  inert  certain  infective  bacteria  that  enter  the 
alimentary  tract  by  way  of  the  mouth.  Of  all  the  defenses, 
however,  none  are  certainly  of  so  much  importance  as  those 
to  be  detected  within  the  internal  structures  of  the  normal 
animal.  In  its  warfare  against  the  invasion  of  infective 
bacteria  and  the  activities  of  their  poisonous  products,  the 
most  significant  defenses  possessed  by  the  body  are  those 
which  directly  aim  at  the  destruction  of  the  living  germs  of 
disease  and  at  the  neutralization  of  their  poisonous  waste 
products. 

In  so  far  as  we  now  know  the  internal  means  of  defense 
used  by  the  body  in  its  warfare  against  infective  bacteria 
and  their  poisonous  products  are  the  phagocytic  cells,  such 
as  the  leukocytes,  the  large  mononuclear  cells  of  the  blood, 
and  the  connective  tissue  and  endothelial  cells,  and  the 
ill-defined  vital  substances  in  the  circulating  blood  which 
act,  so  to  speak,  as  antidotes  to  bacterial  poisons.  If  these 
defenses  are  not  of  sufficient  vigor  to  destroy  the  invading 
bacteria,  or  to  render  inert  the  poisons  produced  by  them, 
the  bacteria  are  victorious  and  infection  results;  on  the 
other  hand,  if  there  be  failure  to  excite  disease,  the  tissues 
have  been  victorious,  and  are  then  said  to  be  resistant  to 
or  immune  from  this  or  that  particular  type  of  infection. 

In  some  cases  the  protective  agents  possessed  by  the 
animal  organism  act  directly  upon  the  invading  parasites 
themselves — i.  e.,  they  are  germicidal;  in  others  their 
function  is  more  that  of  antidotes,  or  neutralizes'  in  the 
chemical  sense,  of  the  poisons  produced  by  these  parasites, 
the  parasites  themselves,  in  certain  instances,  experiencing 
only  slight  injury  from  a  limited  sojourn  in  the  living  tissues. 


THE  DEFENSES  OF  THE  BODY  279 

So  far  as  we  can  learn  the  blood  serum  exhibits  normally 
a  small  amount  of  antitoxic,  agglutinative,  and  bactericidal 
action  against  a  great  variety  of  pathogenic  bacteria.  The 
nature  of  the  agents  responsible  for  these  activities  is  believed 
to  be  identical  with  that  of  similar  agents  found  in  the 
blood  of  artificially  immunized  animals,  though  in  the  latter 
instance  they  are  always  present  to  a  higher  degree  than 
in  normal  animals. 

To  those  ill-defined  substances  whose  affinities  are  re- 
stricted to  the  soluble  toxins  elaborated  by  the  invading 
bacteria  the  name  "antitoxins"  is  now  generally  applied. 
Contrary  to  what  we  have  seen  in  the  case  of  the  germicidal 
substances,  normally  present  in  the  blood,  antitoxins  are 
to  be  detected  in  the  normal  animal  organism  in  very  small 
amounts.  When  they  do  exist  under  such  conditions  they 
are  of  but  comparatively  feeble  potency.1 

In  the  great  majority  of  instances  antitoxic  activities  are 
acquired  peculiarities;  acquired  in  some  cases  in  a  more  or 
less  natural  manner,  as  in  the  course  of  a  non-fatal  attack 
of  a  specific  malady;  induced  in  others  by  purely  artificial 
means,  as  in  the  case  of  immunization  from  diphtheria 
and  tetanus. 

Our  acquaintance  with  the  antitoxins  extends  little  beyond 
their  physiological  functions  and  some  of  .the  means  that 
induce  their  generation.  We  have  no  satisfactory  knowledge 
of  their  intimate  nature  or  of  the  primary  sources  of  their 
production.  They  are  believed  by  some  (Buchner2  and 
Metchnikoff3)  to  represent,  when  artifically  induced,  bac- 

1  See  Bolton,  Transactions  of  Association  of  American  Physicians,  1896, 
xi,  62.     Pfeiffer,  Deutsche  med.  Wochenschrift,  1896,  No.  8.     Fischl  and 
v.    Wanschheim,    Centralblatt    fur    Bakteriologie,'   Parasitenkunde,    und 
Infektionskrankheiten,  1896,  Abt.  i,  Bd.  xix,  S.  652.    Wassermann,  Berliner 
klin.   Wochenschrift,    1898,   No.    1. 

2  Miinchener  med.  Wochenschrift,  1893,  Nos.  24  and  25. 

3  Weil's  Handbuch  der  Hygiene,  Bd.  ix,  Lieferung  1,  S.  48. 


280  BACTERIOLOGY 

terial  toxins  that  have  b'een  modified  by  the  vital  action 
of  the  integral  cells  of  the  body;  and  Roux1  and  Buchner2 
maintain  that  they  exhibit  their  protective  functions  less 
by  direct  combination  with  the  toxins  than  by  a  specific 
stimulation  of  the  tissue-cells  that  enables  the  latter  to 
resist  the  harmful  influences  of  the  toxins.  On  the  other 
hand,  Behring,3  Ehrlich,4  and  their  associates  contend  that 
they  are  vital  tissue  elements,  having  the  property  of  com- 
bining directly  with  the  toxins  to  form  "physiologically 
inert  toxin-antitoxin"  compounds  that  dre  in  a  manner 
analogous  to  the  double  salts  of  familiar  chemical  reactions. 

Natural  Immunity. — It  is  well  known  that  among  man 
and  the  lower  animals  individuals  are  frequently  encountered 
who  are,  in  general,  less  susceptible  to  infection  than  are 
others  of  their  species;  and  that  particular  species  of  animals 
not  only  do  not  suffer  naturally  from  certain  specific  diseases, 
but  resist  all  efforts  to  produce  the  diseases  in  them  by 
artificial  methods;  in  other  words,  they  are  naturally  im- 
mune from  them.  The  term  "natural  immunity,"  as  here 
employed,  implies  a  congenital  condition  of  the  individual 
or  species,  a  condition  peculiar  to  his  idioplasm,  which  has 
been  transmitted  to  him  as  a  tissue-characteristic  through 
generations  of  progenitors. 

Acquired  Immunity. — Again,  it  is  often  observed  that  an 
individual  or  an  animal  after  having  recovered  from  certain 
forms  of  infection  has  thereby  acquired  protection  from 
subsequent  attacks  of  like  character;  in  other  words,  they 
are  said  to  have  acquired  immunity  from  this  disease.  "  Ac- 

1Annales  de  1'Institut  Pasteur,  1894,  p.  722. 
2  Berliner  klin.  Wochenschrift,   1894,  No.  4. 
8  Infektion  und  Disinfektion,  Leipzig,  1894,  S.  248. 

<Klinisches  Jahrbuch,  1897,  Bd.  vi,  Heft  2,  S.  311.  Fortschritte  der 
Medicin,  1897,  Bd.  xv,  No.  2. 


THE  DEFENSES  OF  THE  BODY  281 

quired  immunity"  implies,  therefore,  a  condition  of  the 
tissues  of  an  individual,  not  of  necessity  peculiar  to  other 
members  of  the  race  or  species,  that  has  originated  during 
his  life  from  the  stimulation  of  his  integral  cells  by  one  or 
another  of  the  specific  infective  irritants  that  may  have 
been  purposely  introduced,  or  accidentally  gained  access 
to  his  body.  Acquired  immunity  may  be  either .  active  or 
passive  in  character. 

Active  Immunity. — Active  immunity  is  that  seen  after 
recovery  from  infection  acquired  in  a  natural  way,  or  from 
infection  induced  by  the  injection  of  dead  or  living  organisms 
or  the  poisons  peculiar  to  them. 

Passive  Immunity. — Passive  immunity  is  that  condition 
in  which  protective  substances  that  have  been  generated  in 
a  susceptible  animal  by  one  or  the  other  methods  of  active 
immunization  are  transferred  directly  from  that  animal  to 
•a  normal  animal  by  the  injection  of  the  blood  serum  of  the 
former  into  the  tissues  of  the  latter;  the  latter  being  as  a 
rule  at  once  protected.  The  antitoxic  serums  have  been 
employed  most  frequently  to  bring  about  passive  immunity. 
The  protective  value  of  diphtheria  antitoxin  in  those  that 
have  been  exposed  to  infection  is  well  established.  The  use 
of  tetanus  antitoxin  for  prophylactic  purposes  is  also  recom- 
mended in  cases  where  there  is  a  possibility  of  the  develop- 
ment of  tetanus. 

Vaccination  Against  Bacterial  Diseases. — The  employment 
of  various  prophylactic  vaccinations  against  infectious  dis- 
eases has  received  much  attention  in  recent  years.  The 
measures  employed  in  different  diseases  vary  somewhat, 
though  in  general  the  principles  are  similar. 

The  first  measures  of  this  nature  that  were  employed  on 
a  large  scale  are  those  of  Haffkine  in  vaccination  against 


282  BACTERIOLOGY 

cholera  and  plague  by  means  of  cultures  that  had  been 
killed  by  heating  to  a  moderate  temperature.  Such  dead 
organisms  when  injected  bring  about  a  reaction  in  the  body 
which  is  manifested  by  a  marked  increase  in  the  specific 
agglutinative  and  bactericidal  properties  of  the  blood- 
serum. 

Wright  introduced  a  similar  method  of  vaccination  against 
typhoid  fever.  The  prophylactic  treatment  consists  of  one 
or  more  injections  of  dead  cultures  of  bacillus  typhosus.1 

Metchinkoff  and  Besredka  maintain  that  immunity  is 
less  complete  and  is  accompanied  by  more  severe  reactions 
when  induced  by  dead  bacterial  vaccines  than  when  a  small 
quantity  of  "sensitized"  living  culture  is  employed. 

With  this  in  mind  they  prepare  vaccines  by  subjecting 
living  cultures  to  the  action  of  specific  immune  serum  that 
has  been  heated  sufficiently  to  destroy  its  disintegrating 
power.  By  this  plan  the  haptophore  side  chains  of  the  bac- 
teria are  saturated  with  specific  immune  bodies,  manifested 
by  the  agglutination  of  the  bacteria.  The  agglutinated  mass 
is  then  washed  to  remove  the  serum,  centrifuged  and  the 
sediment  used  for  vaccination.  The  subcutaneous  injection 
of  vaccines  so  prepared  is  said  to  be  followed  by  little  or 
no  local  pain  and  almost  no  constitutional  reactions.  These 
advantages  are  attributed  to  the  sensitization  in  vitro, 
which  would  otherwise  go  on  within  the  tissues  and  account 
for  the  undesirable  reactions. 

The  method  of  Gay  differs  from  the  foregoing  in  that  the 
sensitized  living  bacteria  are  killed  by  heating  before  they 
are  injected. 

1  See  chapter  on  Typhoid  Fever. 


THE  DEFENSES  OF  THE  BODY  283 

Precipitins. — The  immunization  of  animals  with  a  variety 
of  substances  other  than  bacteria  has  served  further  to  de- 
monstrate the  complex  mechanism  of  immunity.  One  of  the 
reactions  that  is  noticed  as  the  result  of  such  immunization 
is  the  precipitation  observed  when  the  serum  of  the  immu- 
nized animal  is  mixed  with  the  substance  with  which  it  has 
been  treated.  For  instance,  the  serum  of  an  animal  that 
has  received  repeated  injections  of  blood,  tissue  juices  or 
certain  secretions  from  alien  species,  will  cause  a  precipi- 
tate to  form  when  mixed  with  either  of  these  substances 
in  vitro.  These  "precipitins,"  as  the  newly  formed  bodies 
in  the  blood  of  the  treated  animal  are  called,  are  specific 
in  that  they  form  precipitates  only  with  the  materials 
injected. 

This  precipitin  reaction  is  so  characteristic  that  it  is 
employed  for  the  identification  of  blood  in  medico-legal 
cases  requiring  the  differentiation  between  human  blood 
and  that  of  domestic  animals;  thus,  the  serum  of  a  rabbit 
into  which  human  blood  has  been  injected  will  cause  a 
precipitate  with  no  other  blood  except  that  of  the  anthro- 
poid ape. 

In  like  manner,  the  repeated  injection  of  milk  of  one 
species  of  animal  into  the  tissues  of  another  will  result  in 
the  formation  of  specific  precipitins  in  the  blood  serum  of 
the  treated  animal,  that  will  precipitate  only  the  milk  of 
that  species  of  animal  from  which  the  milk  was  derived. 

Agglutinins. — If  the  blood  serum  of  an  individual  who  has 
recovered  from  a  bacterial  infection  or  who  has  been  ren- 
dered immune  by  bacterial  vaccination  be  mixed  with  the 
bacteria  that  caused  the  infection  or  those  used  in  the  vac- 
cination— the  bacteria,  if  motile,  lose  their  motility  and 
finally  clump  together  in  masses,  i.  e.,  they  are  "agglu- 


284  BACTERIOLOGY 

tinated"  by  the  serum;  the  reaction  being  referable  to  the 
presence  of  a  new  body — "agglutinin" — that  has  appeared 
in  the  blood  as  a  result  of  the  infection  or  the  vaccination. 
The  relation  of  this  newly  formed  antibody  is  specific,  i.  e., 
it  agglutinates  only  those  agents  that  called  it  forth.  In 
the  normal  blood  agglutinating  activity  may  often  be  de- 
monstrated for  a  variety  of  bacteria  (Bergey)  but  it  is  never 
as  high  in  potency  as  is  that  which  may  be  artificially  induced, 
or  that  seen  early  in  convalescence  from  a  number  of  infec- 
tions. 

The  agglutinating  properties  of  an  immune  serum  are  not 
indicative  of  the  degree  of  immunity  possessed  by  the  indi- 
vidual from  whom  the  blood  was  drawn.  There  may  be  a 
relatively  high  degree  of  agglutinating  property  with  no 
demonstrable  correspondence  in  germicidal  or  protective 
activity.  Though  no  parallelism  necessarily  exists  between 
the  degree  of  agglutinating  and  that  of  germicidal  or  bac- 
teriolytic  activities  of  an  immune  serum,  it  is  nevertheless 
true  that  both  qualities  develop  as  a  result  of  an  effort  on 
the  part  of  the  tissues  to  resist  infection,  and  both  may 
represent  a  response  to  the  same  stimulus. 

The  specificity  of  the  agglutinating  reaction  has  proved 
of  use  in  the  identification  of  infective  bacteria,  and  con- 
versely, in  the  recognization  of  diseases  resulting  from 
bacterial  invasion.  For  instance:  given  an  unidentified 
bacterium  of  the  colon — typhoid — dysentery  group  that 
is  agglutinated  by  the  serum  from  a  case  either  of  experi- 
mentally induced  or  naturally  acquired  typhoid  fever  and 
is  not  agglutinated  by  serum  from  a  dysentery  case  or  one 
of  colon  infection — in  all  human  probability  that  organism 
is  the  typhoid  bacillus;  or  given  the  serum  from  a  patient 
suffering  from  an  undetermined  febrile  disease  that  agglu- 


THE  DEFENSES  OF  THE  BODY  285 

tinates  Bacillus  typhosus  and  no  other  organism,  that  patient 
in  all  probability  is  suffering  from  typhoid  fever.  This 
latter  application  of  the  reaction  constitutes  what  is  gener- 
ally known  as  the  Widal  reaction. 

Immunity:  Historic  Sketch. — In  the  course  of  our  studies 
aimed  to  secure  light  on  the  mechanism  of  infection,  two 
phenomena  are  constantly  in  evidence,  notably — first, 
that  not  all  individuals  are  susceptible  to  infection  by  all 
pathogenic  bacteria,  and  next,  that  an  individual  who  has 
recovered  from  infection  has  undergone  a  change  during 
the  course  of  the  disease  that,  as  a  rule,  renders  him 
insusceptible  to  subsequent  infection  by  the  same  species 
of  bacteria.  Individuals  in  either  the  one  or  the  other 
state  are  said  to  be  immune;  in  the  former  to  be  immune  by 
nature,  in  the  latter  to  have  acquired  immunity. 

In  its  present  development  there  is  no  more  fascinating 
subject,  and  none  of  broader  biological  significance  than 
that  involving  this  riddle  of  immunity.  For  a  quarter  of 
a  century  it  has  attracted  the  attention  of  the  most  brilliant 
investigators  in  medicine  and  its  cognate  fields,  and,  though 
much  has  been  learned,  it  is  as  yet  far  from  fully  elucidated. 
It  is  obviously  inadvisable  in  a  work  of  this  character  to 
follow  in  detail  the  manifold  lines  of  investigation  aimed  to 
clear  up  this  matter.  We  shall  content  ourselves,  therefore, 
with  a  statement  of  the  significant  results  and  such  discus- 
sion of  them  as  may  be  necessary  to  indicate  their  bearings 
upon  the  problem. 

Knowing  as  we  now  do  that  infection  is  at  bottom  a 
matter  of  intoxication,  and  believing,  as  we  are  led  to 
do  by  Ehrlich  and  his  pupils,  that  intoxication  is  to  be 
interpreted  as  a  destructive  union,  in  the  chemical  sense, 
between  the  poisons  on  the  one  hand  and  cells  or  parts 


286  BACTERIOLOGY 

of  cells  for  which  they  have  an  affinity  on  the  other, 
natural  resistance  or  immunity  from  one  or  another  type 
of  infective  organism  may  be  interpreted  in  several  ways, 
namely — that  the  naturally  immune  animal  is  by  nature 
devoid  of  those  cells  or  parts  of  cells  for  which  the  poison 
of  the  infective  organism,  from  which  it  is  immune,  has  a 
specific  destructive  affinity;  or,  that  the  animal  is  by  nature 
endowed  with  cells,  parts  of  cells  or  products  of  cell  life  that 
serve  as  antidotes  for  the  poison  of  the  infective  organism 
in  question;  or,  again,  that  certain  cells  of  the  immune 
animal  have  the  power  to  actually  destroy  the  infective 
organism  when  it  gains  access  to  the  body,  thereby  not  only 
preventing  its  growth  and  multiplication,  but  simultaneously 
rendering  inert  the  poisons  liberated  as  a  result  of  its 
disintegration. 

Long  before  the  present  state  of  our  knowledge  on  this 
subject  had  been  reached,  observers  who  were  occupied 
with  the  study  of  infection  had'  offered  certain  explanations 
for  the  occasional  failure  of  their  efforts  to  cause  disease  by 
inoculation.  In  the  majority  of  cases  such  doctrines  or 
hypotheses  were  offered  in  connection  with  the  immunity 
that  had  been  acquired.  This  is  not  surprising,  since  artifi- 
cially induced  immunity — L  e.,  acquired  immunity — is  a 
constitutional  state  that  is  more  or  less  under  the  control 
of  the  experimentor,  while  natural  immunity  is  an  heredi- 
tary, idioplasmic  peculiarity  that  can  be  modified  little  if 
at  all  by  any  of  the  known  experimental  procedures. 

Among  the  first  to  offer  an  explanation  for  the  condition 
of  acquired  immunity  was  Chauveau,  who,  in  1880,  sug- 
gested that  the  immunity  commonly  observed  in  animals 
that  had  recovered  from  a  specific  infection,  and  likewise 
immunity  produced  artificially  by  vaccination,  is  referable 


ELIE   METCHNIKOFF 
1845-1916 


THE  DEFENSES  OF  THE  BODY  287 

to  a  product  of  the  infective  organisms  that  is  retained  in 
the  tissues,  and  which,  by  its  presence  serves  to  prevent 
the  development  of  the  same  species  of  organisms  should 
they  subsequently  gain  access  to  the  tissues.  This  doctrine 
is  usually  known  as  Chauveau's  "Retention  Hypothesis  of 
Immunity."  We  shall  see  later  that  it  is  only  in  small  part, 
if  at  all,  a  tenable  theory. 

As  opposed  to  Chauveau,  Pasteur  and  his  pupils,  in  the 
same  year  (1880),  expressed  the  opinion  that  acquired 
immunity  was  to  be  explained  in  just  the  reverse  way  to 
that  conceived  by  Chauveau.  They  believed  that  in  the 
primary  attack  of  infection  something  was  extracted  from 
the  tissues  by  the  infecting  organisms  that  was  necessary  to 
support  the  growth  of  the  same  species  should  it  subse- 
quently invade  the  body.  This  doctrine  is  known  as  Pas- 
teur's "Exhaustion  Hypothesis"  of  Immunity,  and  has 
apparently  little  claim  to  serious  consideration. 

Four  years  later  (1884)  Metchnikoff,  while  engaged  upon 
the  study  of  certain  lower  forms  of  animal  life,  noticed 
that  particular  mesodermal  cells,  in  the  course  of  their 
wanderings  through  the  body,  had  the  power  to  actually 
pick  up  insoluble  particles  that  had  gained  access  to  it  in 
one  way  or  another.  He  looked  upon  them  as  functioning, 
therefore,  as  scavengers.  These  phagocytes,  as  they  are 
now  generally  known,  are  common  not  alone  to  the  lower 
forms  of  life,  but  to  the  most  highly  organized  as  well.  In 
the  higher  forms  of  animal  life,  the  function  of  phagocytosis 
is  conspicuously  exhibited  by  the  wandering  cells— i.  e., 
the  white  blood  corpuscles.  In  a  lower  degree  the  inclusion 
of  foreign  bodies  with  their  subsequent  digestion  or  disin- 
tegration may  occasionally  be  seen  in  other  cells  as  well. 

Metchnikoff  believed  this  phagocytic  power  to  be  the 


288  BACTERIOLOGY 

most  important  defensive  mechanism  possessed  by  the  body, 
and  believed  both  natural  and  acquired  immunity  to  be 
referable  to  it;  in  the  former  case  regarding  it  as  a  natural 
endowment,  in  the  latter  as  a  function  that  had  been  excited 
by  the  specific  stimulus  offered  by  the  organisms  or  their 
poisons  that  were  concerned  in  the  primary  attack  of  disease 
from  which  the  animal  recovered,  or  by  the  organisms  used 
in  purposely  exciting  a  modified  form  of  the  disease  by  one 
or  another  of  the  modes  of  protective  vaccination. 

As  the  phenomenon  of  phagocytosis  could  easily  be  ob- 
served under  the  microscope,  and  its  observation  therefore 
accessible  to  all  interested  in  the  question,  the  plausibility 
of  the  doctrine  at  once  attracted  many  adherents,  and 
Metchnikoff  s  views  were  everywhere  accepted  as  the  prob- 
able explanation  of  the  defensive  mechanism  of  the  body 
against  infection. 

In  a  little  while,  however,  Fluegge,  of  Breslau,  perceiving 
the  incompetency  of  both  Chauveau's  and  Pasteur's  doc- 
trines, observing  occasional  inconsistencies  in  Metchnikoff  s 
teaching,  and  recalling  certain  significant  reactions  of  the 
blood  that  had  appeared  in  the  course  of  experiments  by 
Traube  and  Gscheidlen,  by  Fodor,  by  Rauschenbach,  and 
by  Grohmann,  determined  to  subject  the  whole  question 
to  an  experimental  critical  review. 

To  Nuttall,  an  American  working  in  his  laboratory,  was 
assigned  the  question  of  determining  if  the  cell-free  blood, 
or  the  plasma,  was,  as  had  been  suggested  by  Grohmann, 
possessed  of  germ-destroying  properties.  Nuttall's  work 
resulted  in  a  blow  to  MetchnikofFs  doctrine  that  for  a  long 
time  seemed  to  be  fatal.  He  demonstrated  that  certain 
virulent  bacteria  were  rendered  incapable  of  development, 
incapable  of  infecting  susceptible  animals,  and,  in  short, 


THE  DEFENSES  OF  THE  BODY  289 

killed  by  exposure  to  the  serum  of  animal  blood  free  of  all 
cellular  elements.  These  results  naturally  caused  defections 
from  the  ranks  of  MetchnikofFs  followers,  especially  since 
Nuttall's  deductions  were  fully  confirmed  by  many  dis- 
tinguished experimenters.  In  consequence,  for  a  number  of 
years  after  Nuttall's  work,  the  cell-free  fluids  of  the  body 
were  regarded  as  the  real  defenses  of  the  body  in  so  far  as 
invading  bacteria  were  concerned. 

The  natural  sequel  of  NuttalPs  demonstration  was  a 
general  curiosity  as  to  the  manner  in  which  the  destruction 
of  'bacteria  was  accomplished  by  the  cell-free  serum;  the 
conditions  that  modify  the  phenomenon;  and  the  nature  of 
the  ingredient  of  the  serum  to  which  the  germicidal  activity 
might  properly  be  referred. 

Buchner  demonstrated  that  active  serum  was  robbed  of 
its  germicidal  power  by  dilution  with  water  and  by  dyaliza- 
tion;  that  it  was  not  affected  by  dilution  with  physiological 
salt  solution;  that  it  was  rendered  inert  by  an  exposure  of 
fifty  minutes  to  55°  C.,  and  that  it  was  not  affected  by 
alternate  freezing  and  thawing.  He  concluded  that  the 
element  of  the  blood  to  which  the  function  of  killing  bacteria 
may  be  ascribed  is  a  living  albumen  and  suggested  "alexin" 
as  the  appropriate  designation.  Hankin  and  Martin  believed 
the  active  germicidal  principle  to  be  a  globulin,  a  view  that 
was  to  some  extent  -suggested  by  the  investigations  of  Ogata 
and  of  Tizzoni  and  Cattani;  while  the  investigations  of 
Vaughan  and  of  Kossel  led  them  to  regard  nucleins  as  the 
most  important  constituents  of  the  blood  in  so  far  as  germi- 
cidal action  is  concerned.  Fodor  believed,  as  a  result  of  his 
experiments,  that  the  antibacterial  action  of  the  blood  could 
be  appreciably  accentuated  by  the  addition  of  alkalies. 
While  Baumgarten  and  certain  of  his  pupils  referred  the 
19 


290  BACTERIOLOGY 

death  of  the  bacteria  to  purely  physical  conditions;  believing 
that  their  exposure  to  blood  serum  having  an  osmotic  tension 
different  from  the  fluids  in  which  they  had  been  growing 
resulted  in  disturbances  of  the  bacterial  protoplasm  that 
were  inconsistent  with  bacterial  life. 

By  the  observations  of  Behring  and  Kitasato  and  of  Roux 
and  Yersin  entirely  new  light  was  thrown  upon  the  subjects 
of  infection  and  immunity  and  a  new  field  of  inquiry,  was 
opened.  Through  the  work  of  these  investigators  and  their 
pupils  upon  tetanus  and  diphtheria  it  was  demonstrated 
that  immunity  was,  at  least  in  certain  diseases,  not  so  much 
a  matter  of  actually  destroying  the  invading  bacteria  as  of 
neutralizing  their  poisons. 

The  outcome  of  these  investigations  established  the  fact 
that  if  the  poisons  of  tetanus  bacilli  or  of  diphtheria  bacilli, 
entirely  free  from  the  germs  themselves,  be  injected  into 
susceptible  animals  in  minute  sublethal  doses  the  animals 
presently  acquired  immunity  from  both  poisons  and  living 
organisms.  Furthermore,  that  the  blood  serum  of  animals 
thus  immunized  had  the  power  when  transferred  directly 
to  normal  animals  of  at  once  rendering  them  insusceptible 
to  infection  by  the  living  germs,  and  of  equal  importance, 
that  if  the  blood  serum  of  an  animal  thus  immunized  be 
added  to  the  bacteria-free  poison  of  either  the  tetanus  or 
diphtheria  bacillus  in  a  test-tube  that  the  poison  was  neu- 
tralized, i.  e.j  the  serum  of  the  animal  acted  as  an  antedote 
which  rendered  the  bacteria  poison  inert. 

It  is  obvious  therefore  that  through  the  injections  into 
the  normal  animals  of  non-fatal  quantities  of  the  specific 
bacterial  poisons  the  tissues  had  been  stimulated  to  react 
in  a  manner  quite  in  harmony  with  the  views  of  Buchner 
expressed  in  1883,  to  the  effect  that  the  immunity  seen  in 


THE  DEFENSES  OF  THE  BODY        291 

an  animal  that  has  recovered  from  a  specific  infection  is 
explainable  by  a  "reactive  change"  that  has  occurred  in  the 
tissue  cells,  as  a  result  of  the  primary  infection  or  intoxica- 
tion, which  serves  to  protect  the  animal  from  subsequent 
attacks  of  a  similar  character. 

The  demonstration  that  the  serum  of  an  artificially 
immunized  animal  can  not  only  confer  immunity  upon 
another  animal  but,  in  the  case  of  tetanus  and  diphtheria 
in  particular,  actually  cure  it  after  the  disease  is  in  progress, 
is  one  of  the  most  important  steps  that  has  been  made  in 
this  entire  field  of  inquiry.  The  triumph  resulting  from  the 
practical  application  of  this  principle  to  the  prevention 
and  cure  of  diphtheria  in  man  fairly  marks  an  epoch  in 
modern  medicine.  Though  the  results  attendant  upon  the 
application  of  that  principle  to  the  prevention  and  cure  of 
a  number  of  other  diseases — Asiatic  cholera,  typhoid  fever, 
lobar  pneumonia,  infection  by  the  pyogenic  cocci,  rabies, 
tuberculosis,  plague,  syphilis,  and  snake  bites — have  met 
with  comparatively  indifferent  success,  still  the  knowledge 
gained  through  these  efforts  has  been  of  inestimable  value 
in  stimulating  researaches  that  have  served  to  indicate  not 
only  the  manifold  nature  of  this  complex  problem  but  have 
led  to  discussions  through  which  some  of  its  most  obscure 
•phases  have  been  illuminated. 

Briefly  stated,  the  outcome  favors  the  conclusions  that 
the  mechanism  of  immunity  varies  in  different  diseases,  i.  e., 
that  it  depends  upon  the  specific  peculiarities  of  the  invading 
bacteria.  In  some  instances  it  is  manifested  as  an  effort  on 
the  part  of  the  tissues  to  neutralize  bacterial  poisons,  the 
bacteria  themselves  remaining  unaffected;  in  others  as  an 
actual  destruction,  disintegration  or  digestion  of  the  invad- 
ing bacteria  together  with  the  neutralization  of  such  intra- 


292  BACTERIOLOGY 

cellular  poisons  as  may  be  bound  up  as  integral  portions  of 
their  constituent  protoplasm. 

Furthermore,  in  so  far  at  least  as  induced  immunity  is 
concerned,  the  bulk  of  the  experimental  testimony  supports 
the  opinion  that  the  reaction  is  specific;  that  is  to  say,  be 
the  systemic  reaction  evidenced  as  the  elaboration  of  an 
antidote  to  a  soluble  poison  or  as  increased  facility  to  destroy 
living  bacteria,  it  is  called  forth  only  through  the  specific 
stimulus  afforded  by  the  injection  of  the  animal  with  the 
particular  poison  or  bacterium  from  which  we  desire  to 
protect  it.  Thus,  for  instance,  an  animal  rendered  immune 
from  tetanus  toxin,  is  not  immune  from  diphtheria  toxin 
or  from  the  inroads  of  diphtheria  bacilli;  similarly  an  animal 
immune  from  any  of  the  pathogenic  species  of  bacteria  is 
immune  from  that  species  only  and  not  necessarily  from  any 
others. 

An  observation  of  fundamental  importance  to  an  under- 
standing of  the  mechanism  of  immunity  was  made  by  R. 
Pfeiffer  in  1895.  While  investigating  Asiatic  cholera  he 
found  that  animals  could  be  immunized  from  the  specific 
endotoxin  of  the  organism  causing  that  disease;  that  the 
blood  serum  of  such  immune  animals  when  injected  into 
normal  animals  protected  them  from  what  would  otherwise 
be  a  fatal  d®se  of  the  cholera  spirillum;  that  the  peritoneal 
fluids  of  the  artificially  immunized  animal  had  an  almost 
instantaneous  bacteriolytic,  i.  e.,  disintegrating,  action  upon 
living  cholera  spirilla  that  were  injected  directly  into  the 
peritoneal  cavity;  that  the  serum  from  the  immune  animal 
had  no  such  effect  upon  cholera  spirilla  in  a  test-tube,  but 
if  virulent  cholera  spirilla  were  injected  into  the  peritoneum 
of  an  animal  that  is  not  immune,  and  that  such  injection 
be  followed  immediately  by  an  intraperitoneal  injection  of 


THE  DEFENSES  OF  THE  BODY  293 

blood-serum  from  an  immune  animal,  almost  instantly  the 
peculiar  disintegration  of  the  bacteria  that  was  observed  in 
the  peritoneum  of  the  immune  animal  was  to  be  seen.  As 
we  shall  learn  presently  this  observation  is  of  the  utmost 
importance  and  its  bearing  upon  the  course  of  certain  sub- 
sequent events  will  soon  be  manifest. 

The  significant  features  of  Pfeiffer's  observation  are  that 
while  the  blood  serum  of  an  immune  animal  is  capable  of 
conferring  immunity  upon  a  susceptible  animal,  yet,  in  a 
test-tube  it  exhibits  none  of  the  bacteriolytic  activity  con- 
stantly to  be  noted  in  the  body  of  the  immune  animal;  on 
the  other  hand  if  a  small  quantity  of  it  be  injected  into 
the  peritoneal  cavity  of  a  normal,  susceptible  animal,  the 
phenomenon  of  bacteriolysis,  hitherto  absent,  at  once  makes 
itself  manifest.  Clearly  the  serum  requires  the  cooperation 
of  something  within  the  body  of  the  living  animal  to  bring 
about  the  disintegration  of  bacteria.  The  phenomenon  must 
therefore  be  the  result  of  a  composite  function. 

Though  Nuttall's  work  materially  lessened  the  number 
of  adherents  to  the  phagocytic  doctrine  of  Metchnikoff 
there  was  still  a  group  of  active  workers  who  retained  their 
belief  in  the  fundamental  soundness  of  the  idea.  Metch- 
nikoff himself  never  swerved.  Without  entering  into  a 
discussion  of  the  many  instructive  investigations  upon  the 
questions  of  phagocytosis  it  will  suffice  for  our  purposes  to 
state  briefly  their  culmination.  We  now  know,  through 
the  studies  notably  of  Bail  and  of  Kikuchi  that  on  the  one 
hand  phagocytosis  may  be  inhibited,  and  by  the  demon- 
strations of  Wright  and  Douglass,  in  particular,  that,  on 
the  other,  it  may  be  accentuated.  Bail,  believing  the  real 
defenses  of  the  body  to  be  cellular,  attributes  the  failure 
of  the  cells  to  protect  from  infection  to  an  inhibition  of 


294  BACTERIOLOGY 

their  defensive  powers  by  a  substance,  "aggressin,"  elabo- 
rated by  the  invading  bacteria.  While  Kikuchi,  accepting 
the  "aggressin"  doctrine,  restricts  the  action  of  "aggressin" 
to  the  leukocytes  and  interprets  it  as  in  the  nature  of  a 
negative  chemotactic  phenomenon,  whereby  the  leukocytes 
are  so  repelled  that  they  cannot  approach  and  take  up  the 
bacteria. 

The  efforts  of  Wright  and  Douglass  have  been  in  the  way 
of  accentuating  phagocytic  activity  and  their  results  have 
shed  a  flood  of  most  important  light  upon  the  subject.  In 
1903  and  1904,  in  papers  presented  to  the  Royal  Society 
of  London,  they  express  the  opinion  that  leukocytes  alone 
are  incapable  of  taking  up  bacteria,  and  that  in  order  for 
them  to  exhibit  this  function  the  bacteria  must  first  be 
acted  upon  by  a  something  contained  in  the  normal 
blood,  a  state  of  affairs  analogous  to  that  observed  by 
Pfeiffer.  They  conceived  this  preparation  of  bacteria 
for  ingestion  by  leukocytes  to  be  in  the  nature  of  the  pre- 
paration of  food  for  consumption.  They  employ  the  term 
"Opsonin,"  (meaning  to  cater  for;  to  prepare  food)  in 
designation  of  the  element  in  the  blood  having  that  property. 
Prior  to  the  observations  of  Wright  and  his  associates  it 
had  been  known  that  if  white  blood  cells  be  washed  free  of 
all  adhering  serum  they  are  incapable  of  taking  up  bacteria, 
but  the  interpretation,  in  the  light  of  Wright's  work,  seems 
to  be  incorrect.  It  was  believed  that  a  something  in  the 
blood,  a  "stimulin"  as  it  was  called  by  some,  acted  not  on 
the  bacteria  but  on  the  leukocytes,  stimulating  them  to 
activity.  Wright  and  his  colleagues  have  clearly  shown  the 
error  of  that  view  and  have  convincingly  demonstrated  that 
it  is  the  action  of  their  "opsonin"  on  the  bacteria  that  makes 
phagocytosis  possible.  Thus,  for  instance,  if  bacteria  and 


THE  DEFENSES  OF  THE  BODY  295 

washed  leukocytes  be  brought  together  the  bacteria  are  not 
taken  up  by  the  cells;  if  on  the  other  hand  a  drop  of  normal 
serum  be  added,  phagocytosis  begins.  Or,  if  bacteria  be 
immersed  in  normal  serum  and  then  carefully  cleaned  of  all 
adherent  serum  by  washing  .they  will  readily  be  taken  up 
by  leukocytes,  even  those  also  freed  of  all  serum  by  careful 
washing.  In  short  the  action  of  the  serum  on  the  bacteria, 
through  its  "opsonin,"  has  been  to  make  them  ingestible  or 
digestible  for  the  leukocytes. 

This  opsonizing  property  of  the  blood  varies.  Under 
conditions  depressing  general  health  it  may  be  diminished; 
while  in  the  course  of  infective  diseases  it  is  sometimes 
lessened,  sometimes  increased.  It  may  be  increased  by 
immunization. 

The  nature  of  opsonin  (or  opsonins)  is  not  known.  It  has 
been  suggested  that  they  are  allied  to  the  enzymes.  They 
are  destroyed  by  heat.  They  may  be  absorbed  entirely 
from  the  blood  by  bacteria  with  which  they  combine.  They 
are  unstable,  becoming  gradually  inert  after  withdrawal 
from  the  body. 

In  consequence  of  these  later  investigations  the  phago- 
cytes are  again  to  the  fore  as  one  at  least  of  the  important 
defenses  of  the  body  and  certainly,  in  so  far  as  the  destruction 
of  invading  bacteria  is  concerned,  many  have  come  to  look 
upon  them  as,  after  all,  just  what  Metchnikoff  originally 
regarded  them,  the  true  scavengers  of  the  body. 

Though  the  destruction  of  bacteria  by  the  fluids  of  the 
body  had  been  demonstrated;  though  their  inclusion  and 
digestion  by  phagocytes  could  readily  be  observed;  though 
an  antidote  for  certain  of  their  poisons  could  be  demon- 
strated in  the  blood  of  immunized  animals,  there  was  still 


296  .      BACTERIOLOGY 

wanting  an  explanation  of  the  mechanism  through  which 
these  interesting  phenomena  were  accomplished. 

Omitting  a  group  of  highly  suggestive  observations  made 
by  many  competent  investigators,  we  encounter  the  most 
elaborate  and  at  the  same  time  the  most  fascinating  effort 
to  interpret  the  nature  of  the  reactions  occurring  in  the 
induction  of  immunity  as  well  as  those  fundamentally 
accountable  for  the  natural  condition. 

To  the  genius  of  Ehrlich1  we  owe  the  "side  chain"  or 
"lateral  band"  theory  (Seitenkettentheorie)  of  immunity. 

Its  fundamental  features  comprise  the  acceptance  of 
Weigert's  doctrine  concerning  the  mechanism  of  physiolog- 
ical tissue-equilibrium  and  repair;  and  the  assumption  of  a 
specific  combining  relation,  or  affinity,  between  toxic  sub- 
stances and  the  cells  of  particular  tissues. 

At  the  meeting  of  German  Naturalists  and  Physicians 
held  at  Frankfort-on-the-Main,  in  1896,  Weigert2  advanced 
an  hypothesis  the  essential  features  of  which  are  that 
physiological  structure  and  function  depend  upon  the 
equilibrium  of  the  tissues  maintained  by  virtue  of  mutual 
restraint  between  its  component  cells;  that  destruction  of 
a  single  integer  or  group  of  integers  of  a  tissue  or  a  cell 
removes  a  corresponding  amount  of  restraint  at  the  point 
injured,  and,  therefore,  destroys  equilibrium  and  permits  of 
the  abnormal  exhibition  of  bioplastic  energies  on  the  part 
of  the  remaining  uninjured  components,  which  activity  may 
be  viewed  as  a  compensating  hyperplasia;  that  hyperplasia 
is  not  therefore  the  direct  result  of  external  irritation,  and 
cannot  be,  since  the  action  of  the  irritant  is  destructive  and 


1  Klinisches  Jahrbuch,  1897,  Bd.  vi,  Heft  2,  S.  300. 

2  Neue  Fragestellungen  in  der  pathologischen  Anatomie,  Verhandlungen 
der  Ges.  deutschen  Naturforscher  und  Aerate,  1896,  S.  121. 


PAUL  EHRLICH 
18S4-191S 


THE  DEFENSES  OF  THE  BODY  297 

is  confined  to  the  cells  or  integers  of  cells  that  it  destroys, 
but  occurs  rather  indirectly  as  a  function  of  the  surround- 
ing uninjured  tissues  that  have  been  excited  to  bioplastic 
activity  through  the  removal  of  the  restraint  hitherto 
exerted  by  the  cells  destroyed  by  the  irritant;  and,  finally, 
when  such  bioplastic  activity  is  called  into  play  there  is 
always  fo/percompensation — i.  e.,  there  is  more  plastic 
material  generated  than  is  necessary  to  compensate  for  the 
loss.  Ehrlich  applies  this  idea  to  the  individual  cell,  which 
he  conceives  to  be  a  complex  molecule,  comprising  a  primary 
central  nucleus  to  which  are  attached  by  side  chains  its 
secondary  atom-groups,  in  much  the  same  way  that  our 
conception  of  the  reaction  structure  of  complex  organic 
chemical  compounds  is  represented  graphically.  Injury  to 
one  or  more  of  these  physiologically  essential  atom-groups 
results,  according  to  the  view  of  Weigert,  in  disturbance 
of  the  cell-equilibrium  and  consequent  effort  on  the  part 
of  the  surrounding  atom-groups  at  compensatory  repair. 
With  this  liberation  of  bioplastic  energy  there  is  more 
plastic  material  generated  than  is  necessary  for  the  repair 
of  the  injury.  The  excess  of  this  material  finds  its  way  into  the 
blood  and,  as  we  shall  see  presently,  is  regarded  by  Ehrlich 
as  the  real  antidotal,  immune,  or  protective  substance. 

Assuming  a  specific  combining  relation  between  toxic  sub- 
stances and  particular  cells  or  secondary  atom-groups  of 
cells — and  there  are  experimental  grounds  for  this  assump- 
tion1— it  is  evident  that  the  combination  between  the  intoxi- 
cant and  the  particular  atom-group  for  which  it  has  a  specific 

1  See  Wassermann  und  Takaki,  Ueber  tetanus  antitoxische  Eigen- 
schaften  des  normalen  Centralnervensystems,  Berliner  klin.  Wochen- 
schrift,  1898,  No.  1,  S.  5.  Neisser  und  Wechsberg,  Zeitschrift  fur  Hygiene 
und  Infektionskrankheiten,  Bd.  xxxvi,  S.  299.  Madsen,  ibid.,  Bd.  xxxii,  S. 
214. 


298  .  BACTERIOLOGY 

affinity  is  indirectly  the  cause  of  compensatory  bioplastic 
activity  on  the  part  of  similar  surrounding  atom-groups 
that  have  not  been  destroyed.  This  results,  as  we  learned 
above,  in  hypercompensation,  the  excess  of  plastic  material 
being  disengaged  from  the  parent-cell  and  thrown  free  into 
the  circulating  fluids,  there  to  combine  directly  with  the 
same  intoxicant  should  it  subsequently  gain  access  to  the 
animal.  This  excess  of  plastic  material  thrown  into  the 
circulation  combines,  according  to  Ehrlich,1  directly  with 
the  intoxicant  to  form  physiologically  inactive  "toxin — 
antitoxin' '  compounds,  and  can  therefore  be  reasonably 
regarded  as  the  antitoxic  material  of  animals  rendered 
immune  from  bacterial  and  other  toxins. 

Since  the  announcement  of  that  doctrine  many  important 
advances  have  been  made  in  our  knowledge  of  the  subject. 
We  have  learned  that  the  reactions  of  immunity  or  tolerance 
may  be  induced  by  the  use  of  other  intoxicants  than  those 
elaborated  by  bacteria,  and  by  the  employment  of  other 
cells  and  cell  secretions.  It  has  been  demonstrated  that 
antibodies,  differing  in  their  specific  actions  from  anti- 
toxins, but  originating  probably  in  a  similar  manner,  are 
to  be  detected  in  the  fluids  of  animals  thus  immunized  .or 
rendered  tolerant.  For  a  long  time  we  have  known  of  the 
germicidal  action  of  normal  blood  serum;  since  1895  we 
have  been  familiar  with  the  singular  bacteriolytic  phenome- 
non demonstrated  by  Pfeiffer  in  the  peritoneum  of  animals 
immune  from  cholera;  later  we  learned  that  the  development 
of  immunity  from  a  variety  of  infections  is  accompanied 
by  a  power  on  the  part  of  the  serum  of  the  immune  animal 
to  agglutinate  the  bacteria  causing  the  infection;  the  work 

1  Zur  Kenntniss  der  Antitoxinwirkting,  Fortschritte  der  Medicin,  1897, 
Bd.  xv,  No.  2. 


THE  DEFENSES  OF  THE  BODY  299 

of  Wright  upon  his  opsonic  doctrine  has  finally  placed  the 
leukocyte  among  the  important  defenses  of  the  body  and 
the  profoundly  interesting  investigations  of  Bordet,  Moxter, 
von  Dungern,  Fish,  and  others,  have  shown  that  immunity 
reactions  may  be  induced  with  cells  and  secretions  of  animal 
origin  hitherto  regarded  as  non-irritating  and  harmless.  For 
instance,  we  have  long  known  that  the  blood  of  one  animal 
may  cause  fatal  intoxication  when  injected  into  an  animal 
of  different  species;  but  later  we  learned  if  that  blood  be 
repeatedly  injected  in  non-fatal  amounts,  the  animal  receiv- 
ing the  injections  after  a  while  becomes  tolerant,  and  its 
serum  reveals  the  property  not  only  of  robbing  the  alien 
blood  of  its  hurtful  properties,  but  also  of  actually  dissolv- 
ing its  corpuscles  in  a  test-tube  (hemolysis).  In  an  analogous 
way,  if  such  tissue-cells  as  epithelium  or  spermatozoa  be  in- 
jected repeatedly  into  the  tissues  of  animals,  the  serum  of 
the  blood  of  those  animals  acquires*  the  power  of  agglutinat- 
ing and  finally  dissolving  (digesting)  such  cells  outside  the 
body;  and  if  so  inert  a  secretion. as  milk  be  injected  into 
the  tissues,  the  blood  serum  of  the  animal  receiving  the 
injections  after  a  time  reacts  specifically  with  that  milk  in 
a  test-tube — i.  e.,  precipitates  it. 

From  the  foregoing  we  see  that  in  the  numerous  phases 
and  expressions  of  this  physiological  possibility  there  are 
produced  antibodies  having  functions  totally  different  from 
those  attributed  by  Ehrlich  to  antitoxins — i.  e.,  we  have 
"lysins,"  "  agglutinins,"  "precipitins,"  "  aggressins,"  "op- 
sonins,"  etc.,  that  in  their  mode  of  action  suggest  ferments 
with  specific  affinities.  It  is  evident  that  when  broadly 
conceived  the  mechanism  of  immunity  comprehends  very 
much  more  than  the  neutralization  of  a  bacterial  toxin 
by  an  antitoxin;  and,  what  is  more  to  the  point,  in  many 


300  BACTERIOLOGY 

of  these  conditions  of  immunity  or  tolerance  above  noted 
antitoxins,  as  we  know  them,  are  not  present  at  all. 

In  an  important  series  of  papers  on  the  hemolysins  pub- 
lished by  Ehrlich  and  Morgenroth1  an  effort  is  made  to 
elucidate  further  the  finer  mechanism  of  immunity  in  its 
broad  sense  and  various  expressions,  and  to  adapt  the  side- 
chain  doctrine  to  those  more  complicated  phenomena  in 
which  immunity  depends  not  only  on  the  elaboration  of 
antitoxins,  but  also  upon  a  power  on  the  part  of  the  animal 
fluids  to  cause  a  complete  metamorphosis  or  disappearance 
of  such  particulate  matters  as  bacterial  and  other  irritating 
or  poisonous  cells  and  substances.  They  believe  the  forces 
at  work  in  the  establishment  of  immunity  from  bacteria 
and  from  bacterial  and  other  toxins,  those  operative  in  the 
elaboration  of  the  newly  discovered  lysins,  antilysins, 
agglutinins,  precipitins,  ferments,  antiferments,  etc.,  as 
well  as  those  concerned'  in  physiological  assimilation  and 
nutrition,  to  be  fundamentally  identical.  They  believe 
susceptibility  to  infection,  as  well  as  power  to  assimilate 
nutrition,  to  be  explainable  through  the  assumption  that 
special  molecular  groups  of  the  living  protoplasm  are  endowed 
with  specific  combining  affinities  for  particular  matters;  and 
in  so  far  as  the  establishment  of  disease  is  concerned,  they 
regard  the  receptivity  of  the  individual  to  be  determined 
entirely  by  the  greater  or  less  susceptibility  of  those  pro- 
toplasmic molecular  groups — "receptors,"  as  they  designate 
them — to  disease-producing  agents.  In  individuals  that 
have  been  artificially  immunized  from  hurtful  substances 
they  believe  (in  reiteration  of  Ehrlich's  view  expressed 

1  Berliner  klinische  Wochenschrift,  1899,  Bd.  xxxvi,  S.  6  and  481 ;  1900, 
Bd.  xxxvii,  S.  458  and  681;  1901,  Bd.  xxxviii,  S.  251,  569,  598.  See  also 
Schlussbetrachtung:  Ehrlich  in  Nothnagel's  Speciellen  Pathologic  und 
Therapie,  Bd.  vii,  Theil  1,  Heft  3,  S.  161. 


THE  DEFENSES  OF  THE  BODY  301 

above)  that  the  receptive  molecules  have  been  more  or  less 
multiplied,  according  to  the  degree  of  immunity,  through 
bioplastic  activity  of  similar,  unimpaired  atom-groups 
surrounding  those  more  directly  influenced  by  the  intoxicant 
during  the  process  of  immunization;  and  that  this  excess 
of  such  "receptors/'  although  physiologically  useless,  being 
of  no  known  service  to  normal  function,  circulates  unchanged 
in  the  blood,  and  serves,  through  specific  combining  affinity 
for  the  poison  against  which  the  animal  has  been  rendered 
immune,  to  protect  the  normal  tissues  from  its  hurtful 
action. 

According  to  the  nature  of  the  irritant  from  which  the 
animal  has  been  immunized,  the  "  receptor' '  is  conceived 
to  be  either  of  simple  or  complex  construction,  and  its  pro- 
tective function  to  be  performed  in  either  a  comparatively 
simple  and  direct  way,  or  in  a  more  or  less  complicated  and 
roundabout  manner. 

As  a  result  of  his  studies  of  toxins,  Ehrlich  reached  the 
conclusion  that  they  are  composed  of  at  least  two  function- 
ally distinct  atom-groups:  the  one,  a  "haptophore"  group, 
characterized  by  its  combining  tendencies;  the  other,  a 
"toxophore"  group,  distinguished  for  its  intoxicating  powers; 
and  that  for  the  exhibition  of  its  hurtful  characteristics  a 
toxin  molecule  needs  to  be  first  anchored,  so  to  speak,  to 
the  susceptible  tissue  by  the  "haptophore"  group,  after 
which  its  intoxicating  characteristics  are  exhibited  by  the 
"toxophore"  group.  He  conceives  the  "receptors"  to  be 
likewise  provided  with  "haptophore"  groups  that  pair  with 
the  corresponding  "  haptophores"  of  the  poison  to  which 
the  animal  is  susceptible  or  from  which  it  has  been  immu- 
nized. Where  immunization  has  been  induced  against  such 
relatively  simple  substances  as  toxins,  ferments,  and  certain 


302  BACTERIOLOGY 

cell  secretions,  the  "receptors"  and  their  functions  are  com- 
paratively simple — i.  e.,  the  single  haptophore  of  the  simple 
receptor  pairs  with  that  of  the  intoxicant  and  a  physiologi- 
cally inert  complex  results.  He  conceives  antitoxins  to  be 
simple  receptors  of  this  type,  and  believes  the  neutralization 
of  toxins  by  them  to  take  place  in  this  manner.  On  the 
other  hand,  if  the  immunization  of  an  animal  is  accompanied 
by  an  acquired  power  on  the  part  of  its  serum  to  disintegrate 
bacteria,  to  dissolve  alien  erythrocytes,  to  digest  such  cel- 
lular elements  as  epithelium  and  spermatozoa,  to  precipitate 
milk,  or  agglutinate  bacterial  or  blood-cells,  as  the  studies 
of  Pfeiffer,  Bordet,  von  Dungern,  Moxter,  Fish,  Belfonte 
and  Carbon,  Metchnikoff,  Gruber,  Durham,  Widal,  and 
others  have  demonstrated,  then  the  process  becomes  less 
simple,  and  the  atomic  grouping  of  the  receptive  molecule 
is  correspondingly  more  complex.  In  some  cases  the  recep- 
tor is  provided  with  both  a  haptophore  and  a  ferment-like 
(zymophore)  group;  the  function  of  the  former  being  to 
combine  with  and  hold  in  close  proximity  to  the  latter  the 
albumin  molecule  that  is  to  be  destroyed  or  assimilated;  in 
this  way  bringing  and  holding  the  albumin  molecule  directly 
under  the  influence  of  the  zymophore  group.  In  other  cases 
the  "receptor"  functions  symbolically,  so  to  speak,  with 
a  complementary  something  that  circulates  normally  in  the 
blood,  the  so-called  "complement"  of  Ehrlich  and  Mor- 
genroth.  Under  these  circumstances  the  "receptor"  is 
conceived  to  be  provided  with  two  "haptophore"  groups, 
and  becomes  an  "amboceptor,"  therefore,  the  one  hapto- 
phore of  which  takes  up  and  fixes  the  invading  bacteria, 
tissue-cell,  or  albumin  molecule,  while  the  other  pairs  with 
the  corresponding  haptophore  of  the  complement,  fixing 
the  latter  in  close  proximity  to  the  invading  body,  and 


Fig.  S2 

Receptors  of  the  1st  order  (Ehrlich) 


Fig.  ' — Normal  cell,  with 
receptive  molecular  groups, 
gen 


multiplicity      of 
Attacking    anti- 


Fig.  2 — Cell  attacked  at  its  specifically  recep- 
tive point  by  toxin  molecule  (    'I  I  )• 


Fig.  3 — Cell  equilibrium  destroyed  by  com- 
bination of  toxin  molecule  with  specific  re- 
ceptor (O-H-). 


Fig.  4 — Cell  in  hypercompensation  in  effort  to 
repair  injury  indicated  in  3.  Specific  receptive 
molecules  produced  in  excess  (^—).  All  not 
required  for  repair  float  free  in  the  blood  as 
antibodies — in  this  ease  as  antitoxin*  "*  , 


Fig.    5 — Cell     repaired.        Antibodies    floating 
free.     One  combined  \vith  toxin. 


Fig.  53 

Receptors  of  the  2d  order  (Ehrlich). 


Fig.  1 — Normal  cell,  with  multiple  specifi- 
cally receptive  molecular  groups.  Attacking 
antigen  (— ^). 


Fig.  2 — Cell  attacked  at  specifically  recep- 
tive point  by  antigen  (— «4).  This  may  be 
bacteria,  bacterial  extractives,  or  proteins, 
formed  or  amorphous.  They  stimulate  the  pro- 
duction of  agglutinins,  eoagulins,  precipitins, 
etc. 


Fig.  3 — Cell  with  equilibrium  disturbed  by 
union  of  receptive  cell*  molecule  \vith  its 
specific  antigen  /  ErO\  • 


Fig.   4 — Cell   in    hypercompensation    in   effort 
toward    repair.     Excess     of    bioplastie     matter 
This    represents  the    antibodies: 


preeipitins,  agglutinins,  etc.,  of  this  order. 


Fig.  5 — Cell  restored.  Excess  of  antibodies 
float  free.  One  seen  in  combination  with  its 
antigen. 


Fig.  54 

Receptors  of  the  3d  order  (Ehrlich). 


Fig.  1 — Normal  cell,  -with  multiple  receptive 
molecular  groups.  Attacking  antigen  (~J*)- 
Complement  in  surrounding  fluids  =C.  Note 
that  no  complement  is  fixed  by  the  cell  re- 
eeptors. 


O 


Fig.  2 — Antigen  attached  to  its  specific  affin- 
ity.    Complement  is  at  once  fixed 


(4) 


Fig.  3 — Amboeeptor  with  attached  antigen 
and  complement  disengaged  from  cell,  cell 
equilibrium  thereby  impaired. 


Fig.   4. — Cell   in    hypercompensation    in   effort 
toward    repair.     Excess     of    bioplastic    matter 
((JJM    represents  antibodies  of  this  o.J?d)3^T— ^yiC^h,1 
as  bacteriolysins,    hemolysins,  cytoV/srri^,  ^&tc\    •< 


O 


r^0JL°,o*- 

Fig.    3 — Cell    restored.      Excess    of  antibodies       ^' 
free.     No  complement  engaged,   except  in  the 
ease  of  union  bet\veen  antibody   and  antigen. 


THE  DEFENSES  OF  THE  BODY  303 

thereby  favoring  the  immediate  destructive  activity  of  its 
"zymotoxic"  group. 

It  is  of  importance  to  note  in  connection  with  this  hypothe- 
sis, that  both  "receptors"  and  "complement"  are  present 
in  normal  susceptible,  as  well  as  in  immune  animals,  but 
that  during  immunization  only  the  "receptors"  are  multi- 
plied as  a  result  of  the  specific  stimulation  necessary  to  the 
establishment  of  immunity,  hence  the  commonly  employed 
synonymous  designations:  "immune  bodies"  and  "anti- 
bodies." As  such  bodies  are  generated  during  immuniza- 
tion, the  substance  used  for  the  purpose  is  designated 
"antigen" — i.  e.,  generator  of  antibodies. 

The  Origin  of  Complement. — The -origin  of  complement  is 
a  question  that  is  still  unsolved.  Some  investigators  are 
inclined  to  believe  that  it  is  .derived  from  the  leukocytes. 
This  is  the  opinion  of  Metchnikoff  and  his  associates,  while 
others  believe  that  it  is  derived  from  other  cells  and  organs 
as  well  as  from  the  leukocytes.  Again  other  investigators 
believe  that  it  is  not  derived  from  the  leukocytes  at  all, 
but  from  the  cells  of  certain  organs,  for  instance,  the  spleen 
pancreas,  liver,  and  the  bone  marrow.  It  is  impossible  with 
the  knowledge  at  hand  to  state  definitely  the  origin  of  the 
complement. 

On  the  Specificity  of  Complement. — According  to  Ehrlich 
and  his  pupils  the  term  "complement"  is  to  be  used  generi- 
cally  to  indicate  a  group  of  closely  allied  bodies  differing 
from  one  another  in  that  they  possess  specific  relations  to 
particular  antigens.  By  appropriate  methods  they  claim 
to  have  demonstrated  the  multiplicity  of  complement.  They 
state  that  by  particular  treatment  one  or  more  complement- 
ary bodies  may  be  removed  from  normal  blood  while  others 
remain  in  the  blood  intact;  even  by  such  mechanical  pro- 


304  BACTERIOLOGY 

cedures  as  filtering  through  porcelain  some  complements  are 
held  back  while  others  pass  through  with  the  serum. 

On  the  other  hand,  evidence  afforded  by  the  investigations, 
particularly  of  Buchner,  and  Bordet  and  his  pupils  point  in 
the  opposite  direction  so  insistently  as  to  justify  some  doubt 
of  the  accuracy  of  Ehrlich's  views.  Probably  the  most 
important  evidence  in  favor  of  the  unity  of  complement,  as 
conceived  by  these  investigators,  is  afforded  by  the  every  day 
tests  for  fixation  of  complement  (to  be  described  later).  In 
the  light  of  these  tests  "complement,"  it  seems,  must  be 
nonspecific  in  its  physiological  activities,  therefore  it  is  a 
unit. 

SUMMARY. — According  to  the  nature  of  the  intoxicant 
from  which  the  individual  is  immunized,  the  one  or  the  other 
of  the  structurally  and  functionally  different  types  of  recep- 
tors is  increased — i.  e.,  in  immunity  from  a  simple  toxin  the 
simplest  type  of  receptor,  the  antitoxic,  appears  in  the  blood 
(receptors  of  the  first  order,  Ehrlich);  in  immunity  that  is 
associated  with  agglutinating  or  precipitating  powers  on 
the  part  of  the  blood-serum  receptors  having  a  haptophore 
and  a  zymophore  group  appear  (receptors  of  the  second 
order);  while  in  immunity  from  such  molecular  complexes 
as  blood-,  tissue-,  or  bacterial  cells  there  are  produced 
receptors  of  the  third  order,  which  act  through  their  hapto- 
phore groups  as  intermediate  links  between  the  body  to  be 
destroyed  and  the  normally  present  ferment-like  comple- 
ment that  is  to  bring  about  the  destruction.  For  all  the 
foreign  cellular  irritants  from  which  animals  have  been  im- 
munized, be  they  alien  blood,  tissue-cells,  milk,  or  bacteria, 
there  is  assumed  to  be  circulating  normally  in  the  blood 
"complement"  on  the  one  hand,  and  specific  "receptors" 
on  the  other.  This  idea  of  plurality  for  the  complement 


THE  DEFENSES  OF  THE  BODY  305 

is  apparently  the  vulnerable  point  in  the  argument 
(see  above  ''On  the  Specificity  of  Complement")-  At  all 
events,  it  has  been  vigorously  assailed  by  Bordet  and 
Buchner,  especially,  who  as  said  above,  consider  the 
complement  a  unit,  and  who  do  not  regard  it  as  pos- 
sessed necessarily  of  specific  affinities  beyond  those  com- 
mon to  what  may  be  termed  proteolytic  enzymes  in 
general;  and  Buchner  regards  it  as  nothing  more  than  the 
normally  present  "alexin,"  to  which  he  called  attention 
years  ago,  while  with  equal  warrant  Wright  might  regard  it 
as  the  "opsonin"  on  which  he  has  made  such  instructive 
studies.  Whether  these  objections  be  well  taken  or  not, 
whether  the  doctrine  as  a  whole  can  be  accepted  or  not,  the 
experimental  data  on  which  it  is  based  justify  the  opinion 
that  it  is  the  only  satisfactory  working  hypothesis  that  has 
been  offered  in  explanation  of  the  mechanism  of  what 
Buchner  years  ago  designated  the  "reactive  tissue-changes" 
underlying  the  establisment  of  acquired  immunity.1  Ehr- 
lich's  conception  may  be  graphically  represented  as  follows: 

The  observations  serving  as  the  basis  for  this  doctrine 
have  given  to  the  blood  and  fluids  of  the  body  a  new  and 
peculiar  interest.  According  to  circumstances,  there  may 
be  detected  in  the  blood  and  tissue  juices  a  number  of  mole- 
cular complexes  having  totally  different  functions  and 
affinities,  and  therefore  presumably  different  from  one 
another: 

First,  there  is  normally  present  in  the  blood  serum  of 
practically  all  animals  the  defensive  "alexins"  already 
mentioned. 


1  Justice  cannot  be  done  to  the  beauty  and  ingenuity  of  this  conception 
in  so  brief  a  summary  as  is  appropriate  to  a  text-book.  To  be  appreciated  it 
must  be  read  as  it  came  from  the  authors. 
20 


306  BACTERIOLOGY 

Second,  there  are  the  antitoxins,  found  in  the  blood  of 
animals  artificially  immunized  from  special  sorts  of  intox- 
ication, as  well  as  occasionally  in  the  blood  and  tissues  of 
normal  animals,  the  functions  of  which  are  susceptible  of 
demonstration  outside  the  body  as  well  as  within  the  tissues 
of  the  living  animal. 

Third,  a  body  possessed  of  disintegrating,  bacteriolytic 
powers,  a  bacteriolysin — i.  e.,  having  the  property  of  actually 
dissolving  bacteria,  so  that  the  phenomenon  may  be  observed 
under  the  microscope.  This  phenomenon,  generally  known 
as  "Pfeffer's  Phenomenon/'  is  especially  to  be  seen  within 
the  peritoneum  of  guinea-pigs  that  have  been  rendered  im- 
mune from  Asiatic  cholera  and  from  the  typhoid  and  colon 
infections  and  intoxications.  It  is  not  to  be  confounded 
with  the  ordinary  bactericidal  function  of  the  alexins  that 
is  demonstrable  in  most  normal  serums. 

Fo'urth,  a  body,  the  so-called  "agglutinin"  (Gruber), 
that  was  considered  by  Widal  to  represent  a  "  reaction  of 
infection,"  and  not  of  immunity;  though  now  its  presence  is 
generally  supposed  to  indicate  or  coincide  with  an  effort  on 
the  part  of  the  body  to  resist  infection.  The  presence  of  this 
body  in  a  serum  of  an  animal  is  announced  by  its  peculiar 
influence  on  the  activity  and  arrangement  of  the  particular 
species  of  bacteria  from  which  the  individual  is  immune,  or 
with  which  it  is  infected.  In  the  case  of  typhoid  fever  in 
man,  for  instance,  the  serum  obtained  during  the  early  and 
middle  stages  of  the  disease,  when  mixed  with  fluid  cultures 
or  suspensions  of  the  typhoid  bacillus,  causes  the  bacilli  to 
lose  their  motility  and  to  congregate  (agglutinate)  in  masses 
and  clumps,  a  condition  never  seen  in  normal  cultures  of 
this  organism,  and  practically  never  observed  when  normal 
serum  is  employed  instead  of  the  typhoid  serum.  The 


THE  DEFENSES  OF  THE  BODY  307 

blood  of  animals  artificially  immunized  from  cholera,  pyo- 
cyaneus,  typhoid,  dysentery,  and  colon  infections  also  show 
the  presence  of  "agglutinin."  So  far  as  experience  has  gone, 
this  agglutinating  property  is  manifested  in  the  great  major- 
ity of  cases  only  upon  the  particular  organisms  from  which 
the  animal  supplying  the  serum  is  protected;  that  is  to  say, 
the  relation  is  specific.  In  view  of  the  fact  that  the  power 
of  a  serum  to  agglutinate  bacteria  is  regarded  by  many  as  a 
concomitant  of  infection,  the  exhibition  of  this  property  by 
the  blood  of  immune  animals  may  at  first  sight  appear 
paradoxical.  We  should  not  lose  sight  of  the  fact,  however, 
that  agglutinin  is  presumably  distinct  from  the  other  sub- 
stance concerned  in  immunity,  and  its  presence  in  immune 
animals  may,  therefore,  be  reasonably  explained  as  a  more  or 
less  permanent  result  of  the  "reactions  of  infection"  that  were 
coincident  with  the  primary  stimulations  by  specific  infec- 
tive or  intoxicating  matters  necessary  to  the  establishment 
of  the  condition  of  immunity;  nor  should  we  in  this  con- 
nection lose  sight  of  the  fact  that  its  presence  is  constantly 
to  be  demonstrated  in  typical  cases  of  typhoid  fever,  for 
instance,  that  terminate  fatally,  and  that  have  exhibited 
little  or  no  clinical  signs  of  resistance  at  any  time  during 
their  course. 

Fifth,  there  may  be  demonstrated  in  the  blood  of  animals 
that  have  received  repeated  subcutaneous  injections  of 
milk  a  body — a  "precipitin" — that  causes  a  precipitation 
of  milk.  This  precipitation  represents  apparently  a  specific 
reaction,  for  it  occurs  only  when  the  blood-serum  is  mixed 
with  milk  from  the  species  of  animal  that  supplied  the  milk 
used  for  immunization. 

Sixth,  after  the  repeated  injection  of  blood  or  of  emulsions 
of  tissue-cells  into  the  body  of  an  animal,  there  appear  in 


308  BACTERIOLOGY 

the  blood  of  that  animal  certain  solvents,  or  enzyme-like 
bodies,  "hemolysins,"  "  cytolysins,"  etc.,  that  react  specif- 
ically upon  the  blood  or  tissue-cells  injected,  agglutinating, 
disintegrating,  and  finally  completely  dissolving  them. 
Here,  too,  the  relations  are  specific.  If  a  rabbit,  for  instance, 
be  rendered  tolerant  to  or  immune  from  beef-blood,  its 
serum  dissolves  only  the  red  corpuscles  of  bovines;  if  from 
dog's  blood,  then  only  the  corpuscles  of  the  dog  are  dissolved 
by  the  serum  of  the  rabbit.  Similarly,  if  a  rabbit  be  ren- 
dered tolerant  to  injections  of  emulsions  of  epithelium  cells, 
then  its  serum  dissolves  epithelium  and  not  necessarily 
other  cells.  All  these  reactions  may  be  seen  in  a  test-tube 
or  under  the  microscope. 

Seventh,  if  a  hemolyzing  serum,  prepared  as  indicated 
under  the  sixth  observation,  be  heated  for  a  short  time  to 
54°-56°  C.,  it  at  once  loses  the  hemolytic  function,  but 
regains  it  again  if  a  few  drops  of  serum  from  a  normal 
animal  be  added  to  it.  In  this  phenomenon  of  hemolysis 
Ehrlich's  "receptors  of  the  third  order"  are  assumed  to  be 
concerned;  the  heating,  without  injuring  the  receptors  or 
immune  bodies,  destroys  the  "complement,"  and  thereby 
checks  the  process;  but  the  subsequent  addition  of  nor- 
mal serum  supplies  fresh  "complement,"  and  at  once 
restores  the  combination  necessary  to  the  phenomenon  of 
hemolysis. 

Eighth,  if  blood  containing  a  hemolysin  or  a  cytolysin 
be  repeatedly  injected  into  an  animal,  antibodies — "anti- 
lysins" — are  formed,  and  the  serum  of  the  animal  has  the 
power  of  robbing  a  hemolytic  serum  of  its  hemolyzing  func- 
tion if  mixed  with  it  in  a  test-tube. 

Ninth,  if  normal  blood,  containing  complement,  be 
injected  into  the  same  or  another  species  of  animal,  anti- 


THE  DEFENSES  OF  THE  BODY  309 

complement  is  formed,  which  has  the  property  of  inhibiting 
the  action  of  the  complement. 

Tenth,  if  emulsions  of  dead  bacteria-  be  injected  into 
animals,  the  leukocytes  of  that  animal  may  gain  in  power 
to  take  up  and  destroy  living  bacteria  of  the  same  species, 
a  result  usually  attributed  to  an  increase  in  the  opsonizing 
power  of  the  blood. 

Eleventh,  there  exists  in  the  blood  a  body  to  which  Wright 
has  given  the  name  "opsonin,"  which  has  the  function  of 
so  acting  upon  bacteria  that  they  may  be  taken  up  by 
phagocytes.  This  preparation  of  the  bacteria  by  opsonin 
is  regarded  as  a  prerequisite  to  phagocytosis. 

The  foregoing  sketch  affords  but  an  imperfect  idea  of  the 
vast  amount  of  labor  that  has  been  and  continues  to  be 
expended  upon  this  many-sided,  absorbing  topic.  Of  neces- 
sity many  important  contributions  have  been  omitted,  but 
those  noted  will  serve  to  illustrate  the  lines  along  which  the 
solution  of  the  problem  has  been  approached.  As  a  result 
of  such  investigations,  our  knowledge  upon  infection  and 
immunity  may  be  summarized  as  follows: 

1.  That  infection  may  be  considered  as  a  contest  between 
bacteria  and  living  tissues,  conducted  on  the  part  of  the 
former  by  means  of  the  poisonous  products  of  their  growth, 
and  resisted  by  the  latter  through  the  agency  of  phago- 
cytic  cells  and  the  proteid  bodies  normally  present  in  and 
generated  by  their  integral  cells. 

2.  That  when  infection  occurs  it  may  be  explained  either 
by  the  excess  of  vigor  of  the  bacterial  products  over  the 
antidotal  or  protective  proteids  produced  by  the  tissues,  or 
to  some  cause  that  has  interfered  with  the  normal  activity  of 
of  the  phagocytic  cells  and  production  of  the  protective  bodies. 


310  BACTERIOLOGY 

3.  That  in  the  serum  of  the  normal  circulating  blood  of 
many  animals  there  exists  a  substance  that  is  capable,  out- 
side of  the  body,   of  rendering  inert  certain   pathogenic 
bacteria,   but  which   is,   however,   present  in   such  small 
quantities  as  to  be  ineffective.,  either  for  the  protection  of 
the  animal  or  for  the  cure  of  infection  when  introduced  into 
the  body  of  another  animal  already  infected. 

4.  That  immunity  is  most  frequently  seen  to  follow  the 
introduction  into  the  body  of  the  products  of  growth  of 
bacteria  that  in  one  way  or  another  have  been  modified. 
This  modification  may  be  artificially  produced  in  the  prod- 
ucts of  virulent  organisms,   and  then  introduced  into  the 
tissues  of  the  animal;  or  the  virulent  bacteria  may  be  so 
treated  that  they  are  no  longer  of  full  virulence,  and  when 
introduced  into  the  body  of  the  animal  will  produce  poisons 
of  a  much  less  vigorous  nature  than  would   otherwise   be 
the  case. 

5.  That  immunity  following  the  introduction  of  bacterial 
products  into  the  tissues  is  apparently  due  to  the  formation 
in  the  tissues  of  another  body  or  other  bodies  that  act  as 
antidotes  to  the  poisons,  and  thereby  protect  the  tissues 
from  their  hurtful  effects. ' 

6.  That  this  protecting  proteid  which  is  generated  by  the 
cells  of  the  tissues  need  not  of  necessity  be  antagonistic  to 
the  life  of  the  invading  organisms  themselves,  but  in  some 
cases  must  be  looked  upon  more  as  an  antidote  to  their 
poisonous  products. 

7.  That   immunity,   as   conceived   by   Ehrlich,   may   be 
either  "active"  or  "passive."    According  to  this  interpre- 
tation, it  is  "active''  when  resulting  from  an  ordinary  non- 
fatal  attack  of  infectious  disease;  or  from  a  mild  attack 
of  infection  purposely  induced  through  the  use  of  living 


THE  DEFENSES  OF  THE  BODY        311 

vaccines;  or  from  the  introduction  of  cultures  of  the  bacteria 
that  have  been  killed  by  heat;  or  from  the  gradual  intro- 
duction of  toxins  into  the  tissues  until  a  marked  antitoxic 
state  is  reached.  It  is  "passive"  when  occurring  as  a  result 
of  the  direct  transference  of  the  perfected  immunizing  sub- 
stance from  an  immune  to  a  susceptible  animal,  as  by  the 
injection  of  blood  serum  from  the  former  into  the  latter. 
"Passive  immunity"  is,  in  most  cases,  conferred  at  once, 
without  the  delay  incidental  to  the  usual  modes  of  establish- 
ing "active  immunity."  As  a  rule,  "active"  is  a  more 
lasting  than  "passive"  immunity. 

8.  That  phagocytosis  is  effective  in  warding  off  disease 
in  normal  individuals  only  when  the  defenses  of  the  body 
are  fully  active;  when  the  number  of  invading  bacteria 
is  relatively  small  or  when  the  bacteria  are  possessed  of  low 
aggressive  powers.    It  is  probably  a  secondary  process,  the 
bacteria  being  taken  up  by  the  leukocytes  only  after  having 
been  modified  through  the  opsonizing  activity  of  the  serum 
of  the  blood  and  of  other  fluids  in  the  body. 

9.  That  of  the  hypotheses  advanced  in  explanation  of 
acquired  immunity,  the  one  worthy  of  greatest  confidence 
is  that  which  assumes  immunity  to  be  due  to  reactive 
changes  on  the  part  of  the  tissues  that  result  in  the  formation 
in  these  tissues  of  antitoxic  and   other  antibodies,  which 
circulate  free  in  the  blood,  and  in  a  variety  of  ways  serve 
to  screen  the  tissues  from  the  harmful  effect  of  extraneous 
intoxicants  and  irritants,  in  some  cases  acting  principally 
as    antidotes    to    toxins,    in   others   exhibiting    more  the 
germicidal  (bacteriolytic)  than  the  antitoxic  property. 


CHAPTER  XV. 

Hemolysis — The  Hemolytic  System — Identification  of  Specific  Immune 
Bodies  and  Specific  Antigens  by  Their  Ability  to  Fix  Complement — The 
Wassermann  Reaction — Schematic  Representation  of  Reactions. 


THE   HEMOLYTIC   REACTION. 

THE  term  hemolysis  relates  to  a  phenomenon  through 
which  hemoglobin  is  caused  to  escape  in  solution  from  red 
blood  corpuscles.  The  process  is  also  known  as  "laking." 
It  may  be  brought  about  in  a  number  of  ways — physical, 
chemical,  and  vital.  It  is  with  the  latter  that  we  are  here 
concerned. 

As  a  result  of  the  investigations  of  Landois  we  have  known 
for  a  long  time  that  the  blood  of  one  species  of  animal  often 
exhibits  destructive  action  Upon  the  corpuscles  of  the  blood 
of  an  animal  of  another  species.  He  showed  that  grave 
toxic  symptoms,  sometimes  fatal  results,  follow  upon  the 
introduction  of  the  blood  of  one  species  into  the  veins  of 
another.  The  blood  of  the  dog  is  a  powerful  solvent  for  the 
blood  corpuscles  of  many  other  animals,  while  that  of  the 
horse  and  of  the  rabbit  has  very  little  solvent  action.  The 
corpuscles  of  the  rabbit  are  readily  laked  by  the  blood 
serum  of  a  number  of  other  species  while  those  of  the  cat 
and  the  dog  are  much  more  resistent.  The  corpuscles  of 
the  sheep  and  of  the  rabbit  are  dissolved  by  dog's  serum  in 
a  very  few  minutes. 

Landois'  investigations  explain  in  a  satisfactory  way  the 
(312) 


THE  HEMOLYTIC  REACTION  313 

fatalities  so  often  attendant  upon  the  earlier  practices  of 
transfusion,  when  it  was  customary,  after  a  severe  hemor- 
rhage, in  certain  cases  of  poisoning,  especially  by  carbon 
monoxide,  and  in  certain  pathological  states,  to  transfuse 
the  blood  of  animals  into  the  veins  of  man  for  purposes  of 
resuscitation.  So  convinced  was  Landois  of  the  danger  at- 
tendant upon  the  practice  that  he  states :  the  blood  of  animals 
should  never  be  transfused  into  the  bloodvessels  of  man. 
For  a  somewhat  shorter  time  we  have  known  that  if  such 
toxic  alien  blood  be  injected  into  animals  in  non-fatal  quan- 
tities, that  repeated  injections  of  gradually  increasing  doses 
may  be  made  until  a  condition  develops  in  which  the 
receptive  animal  is  immune  from  the  poisonous  action  of 
the  alien  blood.  When  this  point  is  reached  the  blood  of 
the  immunized  animal  exhibits  specific  reactions  with  the 
alien  blood  that  are  not  only  of  very  great  theoretical  interest, 
but,  as  newer  developments  demonstrate,  are  susceptible 
of  application  to  the  solution  of  other  problems  relating  to 
infection  and  resistance.  Thus,  for  instance,  if  a  portion  of 
the  same  blood  used  in  immunizing  the  animal  be  repeatedly 
washed  in  physiological  salt  solution  until  one  has  nothing 
left  but  red-blood  corpuscles  suspended  in  salt  solution 
and  to  this  there  be  added  a  small  amount  of  the  blood, 
serum  from  the  immune  animal,  and  the  mixture  be  allowed 
to  stand  for  a  little  while  at  body  temperature,  there  will 
be  a  more  or  less  complete  solution  of  hemoglobin  from  the 
washed  corpuscles  and  their  stroma  will  finally  collect  at  the 
bottom  of  the  vessel  as  a  more  or  less  pale  or  colorless  mass. 
If  instead  of  using  in  the  experiment  the  serum  just  as  it 
comes  from  the  immune  animal,  we  heat  it  for  thirty  minutes 
to  55°  C.,  and  then  mix  it  with  the  same  volume  of  washed 
corpuscles  suspended  in  salt  solution,  we  find  that  no  solution 


314  BACTERIOLOGY 

of  hemoglobin  occurs.  But,  if  after  a  reasonable  interval 
of  time  we  now  add  to  the  mixture  in  which  no  hemolysis 
has  occurred,  a  small  amount  of  unheated  normal  (not 
immune)  serum — hemolysis  sets  in  almost  at  once  and  may 
proceed  to  completion.  Obviously  in  washing  the  corpuscles 
and  in  heating  the  serum  we  have  eliminated  a  factor 
necessary  to  hemolysis,  which  factor  is  readily  supplied  by 
a  small  quantity  of  fresh,  unheated  serum  from  a  non-immune 
animal.1 

Equally  obviously,  three  factors  are  concerned  in  this 
reaction:  blood  corpuscles,  a  something  in  the  serum  of  the 
immune  animal  that  is  not  affected  by  heating;  and  a 
something  that  is  destroyed  by  the  heating. 

The  heat-proof  body  is  the  amboceptor  of  the  third  order 
of  Ehrlich  or  the  "immune  body" — or  the  "intermediary 
body"  or  the  "antibody"  as  it  is  severally  called.  The 
heat-sensitive  body  is  the  "complement"  of  Ehrlich  or  the 
"alexin"  of  Buchner  and  Bordet.  The  blood  corpuscles  of 
the  alien  species  represent  the  "antigen" — i.  e.,  the  body 
which  when  injected  into  the  animal  being  immunized  stimu- 
lates or  generates  the  production  of  the  specific  antibodies, 
immune  bodies  or  amboceptors  as  we  may  choose  to  call  them. 

We  have  already  learned  that  amboceptors,  or  antibodies 
of  this  order  are  conceived  by  Ehrlich  to  possess  two  hapto- 
phore  groups;  the  one  having  the  power  to  unite  with  a 
corresponding  haptophore  of  the  "complement,"  the  other 
with  a  corresponding  side  chain  haptophore,  or  combining 
group,  of  the  body  to  be  destroyed — in  this  case,  the  alien 
blood  corpuscles.  When  this  combination  is  complete  the 
complement  by  its  ferment-like  action,  destroys  the  blood 

1  Has  this  any  resemblance  to  the  reaction  known  as  "Pfeiffer's  phe- 
nomenon?" 


THE  HEMOLYTIC  REACTION 


315 


corpuscles  which  have  been  "sensitized"  by  their  union  with 
the  antibodies.  Such  destruction  is  not  possible  until  the 
complement  is  bound  by  means  of  the  intermediary  body  with 
the  other  object — the  blood  corpuscle;  neither  is  such 
destruction  possible  when  complement  is,  as  we  just  saw, 
absent  or  rendered  inert  by  heat  or  otherwise.  In  brief, 
we  have  here  a  "system"  the  integers  of  which  must  all 
be  present  and  in  appropriate  adjustment  before  the  desired 
reaction  occurs.  The  several  factors  and  the  reaction  may  be 
for  convenience  of  visualization  graphically  represented,  thus : 


Complement 


Immune  body 


Immune  body 


FIG.  55 


Factors  present  in  the  serum  of  the  immune 

animal. 
No  reaction,  as  antigen  is  absent. 


FIG.  56 

Factor  present  in  heated  immune  serum. 
No    reaction,    as    both    complement    and 
antigen  are  lacking. 


Immune  body 


Antigen 


FIG.  57 


Factors  present  in  heated  immune  serum 
to  which  antigen  has  been  added.  No 
reaction,  as  complement  has  -been  de- 
stroyed by  the  heating. 


316 


BACTERIOLOGY 

FIG.  58 
Complement 


=  Immune  body 


Antigen 


Factors  present  and  in  combination  in  un- 
heated  immune  serum  to  which  antigen 
has  been  added.  Reaction  complete. 


In  the  hemolytic  system  it  is  obvious,  in  so  far  as  two 
factors  are  concerned,  that  specific  relationship  is  essential 
to  the  reaction.  Thus,  immune  serum  from  an  animal 
immunized  from  sheep's  blood  possesses  amboceptors  specific 
for  the  sheep's  blood  corpuscles  and  none  for  the  corpuscles 
of  other  animals,  so  that  if  to  such  immune  serum  blood  cor- 
puscles other  than  those  of  the  sheep  be  added,  no  hemolysis 
occurs,  even  though  it  may  have  been  conspicuously  active 
for  sheep's  corpuscles. 

The  relationship  of  the  complement  to  the  amboceptor  and 
antigen  is  not  specific.  It  reacts  with  any  or  all  amboceptors 
and  antigens  and  is  present  in  all  mammalian  blood. 

It  must  not  be  forgotten,  as  stated  above,  that  natural 
hemolytic  activity  is  sometimes  exhibited  by  one  blood  for 
another,  consequently,  in  arranging  studies  in  this  field 
this  fact  should  be  borne  in  mind  and  care  exercised  to  con- 
trol all  experiments. 


FIXATION   OF   COMPLEMENT. 

From  the  investigations  of  Bordet  and  Gengou  upon  the 
relations  between  antibodies  and  complement,  methods  have 
been  developed  by  which  it  is  possible  to  detect  very  small 
quantities  of  antibodies  in  fluids  under  question  on  the  one 


FIXATION  OF  COMPLEMENT 


317 


hand,  and  to  identify,  on  the  other  hand,  antigens  whose 
true  nature  may  only  be  suspected. 

The  important  points  brought  out  in  their  fundamental 
experiment  are:  that  complement  is  not  specific  in  its 
affinities  and  that  when  once  fixed  by  an  antibody  to  an 
antigen  the  union  is  not  dissociable.  The  experimental  pro- 
cedures necessary  to  this  demonstration  consisted  in  two  series 
of  mixtures — one  in  which  antibody,  its  antigen  and  comple- 


FIG.  59 
SERIES  I. 


=  Plague  antigen. 


Plague  amboceptor. 


=   Complement. 


The  three  factors 
united  after  a 
time. 


FIG.  60 
SERIES  II. 

=  Plague  antigen. 


=  Non-specific  amboceptor 
of  normal  serum. 


=  Complement. 

Only  two  necessary  fac- 
tors present ;  no  union 
possible. 


Washed  corpuscles  and  inactivated  hemolytic  immune  serum  now  added  to 
each  series. 


ment  were  together,  the  other  identical  in  its  ingredients  save 
for  the  absence  of  antibodies  specific  for  the  antigen  used. 
(Figs.  59  and  60.)  It  is  obvious  that  in  the  first  mixture 
(Fig.  59)  all  factors  necessary  to  the  saturation  of  the 
haptophores  of  the  amboceptor  were  present — therefore, 
complement,  being  one  of  these  factors  was  bound  by  the 
amboceptor  to  the  antigen.  In  the  other  mixture  (Fig.  60) 
this  was  not  possible  as  there  were  no  amboceptors  specific 
for  the  antigen  in  it.  But  to  prove  this  "fixation"  of  com- 


318  BACTERIOLOGY 

plement  in  the  first  mixture:  To  this  end,  after  the  mixtures 
had  stood  for  a  time,  an  incomplete  hemolytic  system  was 
added  to  each  mixture— that  is,  an  amount  of  normal 
washed  blood  corpuscles  and  a  portion  of  inactivated 
immune  serum  otherwise  hemolytic  for  those  corpuscles, 
was  added.  Before  this  addition,  obviously,  no  hemolysis 
could  occur,  because  the  complement  of  the  hemolytic 
serum  had  been  destroyed  by  the  heat  used  for  inactivation. 
But  after  the  addition  hemolysis  did  occur  in  one  tube  but 
not  in  the  other.  It  is  plain  that  complement  necessary 
to  the  phenomenon  of  hemolysis  must  have  been  avail- 
able in  one  of  the  tubes,  If  one  recalls  that  in  the  second 

FIG.  61  FIG.  62 

=  Blood  -corpuscle.  (  J     =  Blood  corpuscle. 

b^?|     =  Hemolytic  amboceptor. 
=  Hemolytic  amboceptor.  K|||j 

Jrjpijk     =   Complement. 

No   hemolysis.      No    complement  Hemolysis.  Free  complement  of 

available;   all  fixed,  as  in  a',  original    mixture    now   bound   by 

hemolytic  amboceptor. 

mixture  no  immune  bodies  or  amboceptors  specifically 
related  to  the  antigen  were  present  it  is  clear  that  the  com- 
plement could  not  have  been  bound  or  fixed.  It  must  have 
remained  free  in  the  serum,  available  for  complementing 
the  action  of  the  hemolytic  amboceptors  and  thereby  hemo- 
lyzing  or  destroying  the  normal  blood  corpuscles  added,  as 
shown  by  the  laking  of  such  corpuscles  in  the  tube.  This, 
in  short,  is  what  occurred.  See  Figs.  61  and  62. 

For  this  particular  test)  Bordet  and  Gengou  used  plague 
antigen  (plague  bacilli);  plague  amboceptors  (present  in 
blood  of  animal  immunized  from  plague) ;  complement  (free 
in  normal  mammalian  blood);  normal  serum  (containing  no 


FIXATION  OF  COMPLEMENT  319 

specific  amboceptors) ;  washed  mammalian  blood  corpuscles 
and  inactivated  immune  serum  hemolytic  for  such  corpuscles 
(such  serum  contains  only  hemolytic  amboceptors,  no 
complement). 

The  application  of  the  principles  involved  in  this  experi- 
ment to  the  solution  of  a  number  of  practical  problems  is 
evident.  For  instance,  we  are  called  upon  to  identify  the 
nature  of  an  obscure  infection,  latent  syphilis,  let  us  say. 
We  know  that  the  blood  in  such  cases  contains  specific 
antibodies  for  the  antigen  (excitor)  of  syphilis.  We  know 
that  the*  excitor  of  syphilis  or  -important  extractives  of  it 
are  present  in  the  organs  of  syphilitic  fetuses,  so  that  the 
antigen  is  easy  to  obtain.  We  know  that  all  normal  mamma- 
lian blood  contains  complement.  If,  therefore,  a  mixture  be 
made  of  syphilitic  antigen,  of  normal  guinea-pig  blood  serum 
and  of  the  patient's  blood  serum,  we  have,  providing  the 
patient  be  syphilitic,  all  the  factors  necessary  to  the  union 
of  complement  to  antigen  by  the  amboceptors  of  the  blood. 
If,  after  it  has  stood  for  a  time,  we  now  add  to  such  a  mixture 
hemolytic  amboceptors  and  red  corpuscles  to  which  such 
amboceptors  are  specifically  related,  we  get  no  hemolysis, 
if  the  patient  be  syphilitic,  for  there  is  no  free  complement 
left  for  the  completion  of  the  hemolytic  system — on  the 
other  hand  if  the  patient  be  not  syphilitic,  his  blood  will 
contain  no  amboceptors  capable  of  binding  complement  and 
syphilitic  antigen  together,  therefore,  there  will  be  free 
complement  available  for  the  hemolytic  system  and  hemolysis 
results.  The  application  of  this  principle  to  the  diagnosis 
of  obscure  syphilis  constitutes  what  is  generally  known  as 
"The  Wassermann  Reaction,"  but  it  is  plain  that  the 
principle  is  susceptible  of  application  to  the  identification 
of  other  latent  infective  processes  as  well.  In  fact  it  is  being 
more  and  more  used  for  that  purpose. 


320 


BACTERIOLOGY 


A  glance  at  the  graphic  representation  of  this  reaction  at 
once  also  suggests  the  means  of  identifying  unknown  but 
suspected  antigens.  Thus,  for  instance,  if  in  both  series  we 
have  the  same  amboceptors  and  complement  but  different 
antigens,  one  being  specifically  related  to  the  amboceptor, 
the  other  not,  plainly  we  will  have  a  result  similar  to  that 
obtained  in  the  first  series  after  the  incomplete  hemolytic 
system  is  added — that  is,  there  will  be  no  hemolysis  in  the 
tube  in  which  antigen  and  amboceptor  are  specifically 
related,  for  here  all  free  complement  will  be  fixed — on  the 
other  hand  in  the  tube  in  which  the  antigen  is  not  so  related 
to  the  amboceptor  complement  cannot  be  so  fixed  and  it, 
therefore,  as  in  the  first  experiment,  remains  free  to  complete 
the  hemolytic  system.  The  reaction  may  be  expressed 
graphically  as  follows: 


a< 


FIG.  63 
SERIES  I. 


=   Plague  antigen. 


=  Plague  amboceptor. 


Complement. 

Factors  united. 


FIG.  64 
SERIES  II. 

=   Unknown  antigen. 


Plague  amboceptor. 


:   Complement. 
No  union  possible. 


Washed  corpuscles  and  inactivated  hemolytic  immune  serum  now  added 
to  each  mixture. 

In  the  second  application  of  this  test  observe  that  the 
unknown  antigen  used  in  Series  II  is  not  of  the  nature  of 


FIXATION  OF  COMPLEMENT  321 

plague  antigen.  Had  the  problem  involved  the  identifica- 
tion of  any  other  antigen — say  gonococci,  bacillus  mallei 
or  others — one  would  substitute  in  the  mixtures  gonorrhea 
antibodies  or  glanders  antibodies  or  others  as  the  case  may 
be,  and  proceed  as  above.  In  these  cases,  however,  such 
antibodies  must  be  artificially  produced  in  animals  that 
react  to  gonorrhea  or  glanders  antigens. 

In  addition  to  the  foregoing  the  principles  involved  in 
these  reactions  have  been  employed  for  the  differentiation 
of  closely  allied  proteins.  Such  for  instance  as  the  differen- 
tiation of  bloods.  For  instance,  if  an  animal  be  immunized 
from  human  blood  its  serum  will  contain  amboceptors  for 
human  blood  corpuscles,  the  antigen.  Such  amboceptors 
in  the  presence  of  human  corpuscles  or  their  protein  extrac- 
tives and  complement  fix  the  complement;  on  the  other 
hand  if  the  blood  under  question  be  from  other  species  than 
man,  no  such  fixation  can  occur  as  there  is  no  specific  affinity 
between  such  blood  and  the  amboceptors  for  human  blood. 
Consequently,  in  the  final  test  for  fixation,  as  determined 
by  +  or  —  hemolysis,  no  hemolysis  occurs  after  the  addition 
of  hemolytic  amboceptors  and  their  related  corpuscles  to 
the  mixture  of  'human  blood,  its  amboceptors  and  comple- 
ment, while  hemolysis  does  occur  in  the  mixture  of  alien 
blood,  human  amboceptors,  and  complement. 

In  the  former  case  all  complement  was  fixed  to  the  antigen 
by  the  homologous  amboceptors,  while  in  the  latter  this  was 
not  possible  because  of  the  lack  of  specific  affinity  of  human 
blood  amboceptors  for  the  alien  blood. 

While  the  principles  involved  in  the  practical  application 
of  these  reactions  are  very  simple,  yet  there  are  so  many 
chances  for  error  that  each  and  every  step  demands  the  most 
careful  control. 
21 


APPLICATION  OF  THE  METHODS  OF 
BACTERIOLOGY. 


CHAPTER  XVI. 

To  Obtain  Material  with  Which  to  Begin  Work. 

EXPOSE  to  the  air  of  an  inhabited  room  a  slice  of  freshly 
steamed  potato  or  a  bit  of  slightly  moistened  bread  upon  a 
plate  for  about  one  hour.  Then  cover  it  with  an  ordinary 
water-glass,  place  it  in  a  warm  spot  (temperature  not  to 
exceed  that  of  the  human  body — 37.5°  C.),  and  allow  it 
to  remain  undisturbed.  In  from  twenty-four  to  thirty-six 
hours  there  will  be  seen  upon  the  cut  surface  of  the  bread 
or  potato  small,  round,  oval,  or  irregularly  round  patches 
which  present  various  appearances.  These  differences  in 
macroscopic  appearance  are  due  in  some  cases  to  the  presence 
or  absence  of  color;  in  others  to  a  higher  or  lower  degree 
of  moisture;  in  some  instances  a  patch  will  be  glistening 
and  smooth,  while  its  neighbor  may  be  dull  and  rough  or 
wrinkled;  here  will  appear  an  island  regularly  round  in 
outline,  and  there  an  area  of  irregular,  ragged  deposit.  All 
these  gross  appearances  are  of  value  in  aiding  us  to  distin- 
guish between  these  colonies — for  colonies  they  are,  and 
under  the  same  conditions  the  organisms  composing  each  of 
them  will  always  produce  growth  of  exactly  the  same  ap- 
pearance. It  was  just  such  an  observation  as  this  that  sug- 

(323) 


324     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

gested  to  Koch  a  means  of  separating  and  isolating  in  pure 
cultures  the  component  individuals  from  mixtures  of  bacteria, 
and  from  it  the  methods  of  cultivation  on  solid  media  were 
evolved. 

If,  without  molesting  these  objects,  we  continue  the 
observations  from  day  to  day,  we  shall  notice  changes  in  the 
colonies,  due  to  the  growth  and  multiplication  of  the  indi- 
viduals composing  them.  In  some  cases  the  colonies  will 
always  retain  their  sharply  cut,  round,  or  oval  outline,  and 
will  increase  but  little  in  size  beyond  that  reached  after 
forty-eight  to  seventy-two  hours;  whereas  others  will 
spread  rapidly  and  quickly  overrun  the  surface  upon  which 
they  are  growing,  and,  indeed,  grow  over  the  smaller,  less 
rapidly  developing  colonies.  In  a  number  of  instances, 
if  the  observation  be  continued  long  enough,  many  of  these 
rapidly  growing  colonies  will,  after  a  time,  lose  their  lustrous 
and  smooth  or  regular  surface  and  will  show  here  and  there 
elevations,  which  will  continue  to  appear  until  the  whole 
surface  becomes  conspicuously  wrinkled.  Again,  bubbles 
may  be  seen  scattered  through  the  colonies.  These  are  due 
to  the  escape  of  gas  resulting  from  fermentation,  which  the 
organisms  bring  about  in  the  medium  upon  which  they  are 
growing.  Sometimes  peculiar  odors  due  to  the  same  cause 
will  be  noticed. 

Note  carefully  all  these  changes  and  appearances,  as  they 
must  be  employed  subsequently  in  identifying  the  individual 
organisms  from  which  each  colony  on  the  medium  has 
developed. 

If  we  now  examine  these  colonies  upon  the  bread  or  potato 
with  a  hand-lens  of  low  magnifying  power,  we  will  be 
enabled  to  detect  differences  not  noticeable  to  the  naked 
eye.  In  a  few  cases  we  may  still  see  nothing  more  than  a 


EXPOSURE  AND  CONTACT  325 

smooth,  non-characteristic  surface;  while  in  others  minute, 
sometimes  regularly  arranged  tiny  corrugations  may  be 
observed.  In  one  colony  they  may  appear  as  tolerably 
regular  lines,  radiating  from  a  central  spot;  and  again  they 
may  appear  as  concentric  rings;  and  if  by  the  methods  which 
have  been  described  we  obtain  from  these  colonies  their 
individual  components  in  pure  culture,  we  shall  see  that 
this  characteristic  arrangement  in  folds,  radii,  or  concentric 
rings,  or  the  production  of  color,  is  characteristic  of  the 
growth  of  the  organism  under  the  conditions  first  observed, 
and  by  a  repetition  of  those  conditions  may  be  reproduced 
at  will. 

So  much  for  the  simplest  naked-eye  experiment  that  can 
be  made  in  bacteriology,  and  which  serves  to  furnish  the 
beginner  with  material  upon  which  to  commence  his  studies. 
It  is  not  necessary  at  this  time  for  him  to  burden  his  mind 
with  names  for  these  organisms;  it  is  sufficient  for  him  to 
recognize  that  they  are  of  different  species,  and  that  they 
possess  characteristics  which  will  enable  him  to  differentiate 
the  one  from  the  other. 

Exposure  and  Contact. — Make  a  number  of  plates  from 
bits  of  silk  used  for  sutures,  after  treating  them  as  follows: 

Place  some  of  the  pieces  (about  5  centimeters  long)  in  a 
sterilized  test-tube,  and  sterilize  them  by  streaming  steam 
for  one  hour  or  in  the  autoclave  for  fifteen  minutes  at  one 
atmosphere  pressure.  At  the  end  of  the  sterilization  remove 
one  piece  with  sterilized  forceps  and  allow  it  to  brush  against 
your  clothing,  then  make  a  plate  from  it;  draw  another 
piece  across  a  dusty  table  and  then  plate  it.  Suspend  three 
or  four  pieces  upon  a  sterilized  wire  hook  and  let  them  hang 
for  twenty  minutes  free  in  the  air,  being  sure  that  they 
touch  nothing  but  the  hook;  then  plate  them  separately. 


326     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

Note  the  results. 

In  what  way  do  these  experiments  differ  and  how  can 
the  differences  be  explained? 

Expose  to  the  air  six  Petri  dishes  into  which  either  sterilized 
gelatin  or  agar-agar  has  been  poured  and  allowed  to  solidify; 
allow  them  to  remain  exposed  for  five,  ten,  fifteen,  twenty, 
twenty-five,  and  thirty  minutes  in  a  room  where  no  one  is 
at  work.  Treat  a  second  set  in  the  same  way  in  a  room  where 
several  persons  are  moving  about.  Be  careful  that  nothing 
touches  them,  and  that  they  are  exposed  only  to  the  air. 
Each  dish  should  be  carefully  labelled  with  the  time  of  its 
exposure. 

Do  they  present  different  results?  What  is  the  reason  for 
this  difference? 

Which  predominate — colonies  resulting  from  the  growth 
of  bacteria,  or  those  from  common  molds? 

How  do  you  account  for  this  condition? 

Sprinkle  a  little  fine  dust  over  the  surface  of  a  plate  of 
sterile  gelatin  or  agar-agar;  examine  the  dust-particles  with 
the  microscope  immediately  after  depositing  them  on  the 
medium,  and  again  after  eighteen  to  twenty-four  hours. 
What  differences  do  you  detect?  What  information  of 
sanitary  importance  does  this  give? 

Under  the  description  of  each  of  the  pathogenic  bacteria 
more  or  less  detailed  directions  will  be  found  for  the  dis- 
covery and  isolation  of  each  of  the  pathogenic  bacteria. 


CHAPTER  XVII. 

Various  Experiments  in  Sterilization  by  Steam  and  by  Hot  Air. 

PLACE  in  one  of  the  openings  in  the  cover  of  the  steam 
sterilizer  an  accurate  thermometer;  when  the  steam  has 
been  streaming  for  a  minute  or  two  the  thermometer  will 
register  100°  C.  Wrap  in  a  bundle  of  towels  or  rags  or  pack 
tightly  in  cotton  a  maximum  (self -registering)  thermometer; 
let  this  thermometer  be  in  the  center  of  a  bundle  large 
enough  to  quite  fill  the  chamber  of  the  sterilizer.  At  the  end 
of  a  few  minutes'  exposure  to  the  streaming  steam  remove 
it;  it  will  be  found  to  indicate  a  temperature  of  100°  C. 

Closer  study  of  the  penetration  of  steam  has  taught  us, 
however,  that  the  temperature  found  at  the  center  of  such 
a  mass  may  sometimes  be  that  of  the  air  in  the  meshes  of 
the  material,  and  not  that  of  steam,  and  for  this  reason  the 
sterilization  at  that  point  may  not  be  complete,  because  hot 
air  at  100°  C.  has  not  the  sterilizing  value  that  steam  has 
at  the  same  temperature.  It  is  necessary,  therefore,  that 
this  air  should  be  expelled  from  the  meshes  of  the  material 
and  its  place  taken  by  the  steam  before  sterilization  is  com- 
plete. This  is  insured  by  allowing  the  steam  to  stream 
through  the  substances  a  few  minutes  before  beginning  to 
calculate  the  time  of  exposure.  There  is  as  yet  no  absolutely 
sure  means  of  saying  that  the  temperature  at  the  center  of 
the  mass  is  that  of  hot  air  or  of  steam,  so  that  the  exact 
length  of  time  that  is  required  for  the  expulsion  of  the  air 
from  the  meshes  of  the  material  cannot  be  given. 

(327) 


328     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

Determine  if  the  maximum  thermometer  indicates  a 
temperature  of  100°  C.  at  the  center  of  a  moist  bundle  in 
the  same  way  as  when  a  dry  bundle  was  employed. 

To  about  50  c.c.  of  bouillon  add  about  1  gram  of  chopped 
hay,  and  allow  it  to  stand  in  a  warm  place  for  twenty-four 
hours.  At  the  end  of  this  time  it  will  be  found  to  contain 
a  great  Variety  of  organisms.  Continue  the  observation, 
and  ultimately  a  pellicle  will  be  seen  to  form  on  the  surface 
of  the  fluid.  This  pellicle  is  made  up  of  rods  which  grow 
as  long  threads  in  parallel  strands.  In  many  of  these  rods 
glistening  spores  will  be  seen.  After  thoroughly  shaking, 
filter  the  mass  through  a  fine  cloth  to  remove  coarser 
particles. 

Pour  into  each  of  several  test-tubes  about  10  c.c.  of  the 
filtrate.  Allow  one  tube  to  remain  undisturbed  in  a  warm 
place.  Place  another  in  the  steam  sterilizer  for  five  minutes; 
a  third  for  ten  minutes;  a  fourth  for  one-half  hour;  a  fifth 
for  one  hour. 

At  the  end  of  each  of  these  exposures  inoculate  a  tube  of 
sterilized  bouillon  from  each  tube.  Likewise  make  a  set 
of  plates  or  Esmarch  tubes  upon  both  gelatin  and  agar-agar 
from  each  tube,  and  note  the  results.  At  the  same  time 
prepare  a  set  of  plates  or  Esmarch  tubes  on  agar-agar  and 
on  gelatin  from  the  tube  which  has  not  been  exposed  to  the 
action  of  the  steam. 

.    The  plates  or  tubes  from  the  unmolested  tube  will  present 
colonies  of  a  variety  of  organisms;  separate  and  study  these. 

Those  from  the  tube  which  has  been  sterilized  for  five 
minutes  will  present  colonies  in  moderate  numbers;  but,  as 
a  rule,  they  will  represent  but  a  single  organism.  Study  this 
organism  in  pure  cultures. 

The  same  may  be  predicted  for  the  tube  which  has  been 


EXPERIMENTS  IN  STERILIZATION  329 

heated  for  ten  minutes,  though  the  colonies  will  be  fewer 
in  number. 

The  thirty-minute  tube  may  or  may  not  give  one  or  two 
colonies  of  the  same  organism. 

The  tube  which  has  been  heated  for  one  hour  is  usually 
sterile. 

The  bouillon  tubes  from  the  first  and  second  tubes  which 
were  heated  will  usually  show  the  presence  of  only  one 
organism — the  bacillus  which  gave  rise  to  the  pellicle- 
formation  in  the  original  mixture.  This  organism  is  bacillus 
subtilis.  It  is  especially  adapted  to  the  study  of  those  various 
degrees  of  resistance  to  heat  that  spore-forming  bacteria 
exhibit  at  different  stages  of  their  development. 

Inoculate  about  100  c.c.  of  sterilized  bouillon  with  a  very 
small  quantity  of  a  pure  culture  of  this  organism,  and  allow 
it  to  stand  in  a  warm  place  for  about  six  hours.  Now  subject 
this  culture  to  the  action  of  steam  for  five  minutes;  it  will 
be  seen  that  sterilization,  as  a  rule,  is  complete. 

Treat  in  the  same  way  a  second  flask  of  bouillon,  inocu- 
lated in  the  same  way  with  the  same  organism,  but  after 
having  stood  in  a  warm  place  for  from  forty-eight  to  seventy- 
two  hours — that  is,  until  spores  have  formed — and  it  will 
be  found  that  sterilization  is  not  complete :  the  spores  of 
this  organism  have  resisted  the  action  of  steam  for  five 
minutes. 

To  determine  if  sterilization  is  complete  always  resort 
to  the  culture  methods,  as  the  macroscopic  and  microscopic 
methods  are  deceptive;  cloudiness  of  the  media  or  the 
presence  of  bacteria  microscopically  does  not  always  signify 
that  organisms  possess  the  property  of  life. 

Inoculate  in  the  same  way  a  third  flask  of  bouillon  with 
a  very  small  drop  from  one  of  the  old  cultures  upon  which 
the  pellicle  has  formed;  mix  it  well  and  subject  it  to  the 


330      APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

action  of  steam  for  two  minutes;  then  place  it  to  one  side 
for  from  twenty  to  twenty-four  hours,  and  again  heat  for 
two  minutes;  allow  it  to  stand  for  another  twenty-four 
hours,  and  repeat  the  process  on  the  third  day.  No  pellicle 
will  be  formed,  and  yet  spores  were  present  in  the  original 
mixture,  and,  as  we  have  seen,  the  spores  of  this  organism 
are  not  killed  by  an  exposure  of  five  minutes  to  steam.  How 
can  this  result  be  accounted  for? 

Saturate  several  pieces  of  cotton  thread,  each  about  2  cm. 
long,  in  the  original  decomposed  bouillon,  and  dry  them 
carefully  at  the  ordinary  temperature  of  the  room;  then  at 
a  little  higher  temperature — about  40°  C. — to  complete 
the  process.  Regulate  the  temperature  of  the  hot-air 
sterilizer  for  about  100°  C.,  and  subject  several  pieces  of 
this  infected  and  dried  thread  to  this  temperature  for  the 
same  lengths  of  time  that  we  exposed  the  same  organisms 
in  bouillon  to  the  steam,  viz.,  five,  ten,  thirty,  and  sixty 
minutes.  At  the  end  of  each  of  these  periods  remove  a  bit 
of  thread,  and  prepare  a  set  of  plates  or  Esmarch  tubes 
from  it.  Are  the  results  analogous  to  those  obtained  when 
steam  was  employed? 

Increase  the  temperature  of  the  dry  sterilizer  and  repeat 
the  process.  Determine  the  temperature  and  time  neces- 
sary for  the  destruction  of  these  organisms  by  dry  heat. 
These  threads  should  not  be  simply  laid  upon  the  bottom 
of  the  sterilizer,  but  should  be  suspended  from  a  glass  rod, 
which  may  be  placed  inside  the  oven,  extending  across  its 
top  from  side  to  side. 

Place  several  of  the  infected  threads  in  the  center  of  a 
bundle  of  rags.  Subject  this  to  a  temperature  necessary  to 
sterilize  the  threads  by  the  dry  method.  Treat  another 
similar  bundle  to  sterilization  by  steam.  In  what  way  do 
the  results  of  the  two  processes  differ? 


CHAPTER  XVIII. 


Methods  of  Testing  Disinfectants  and  Antiseptics — Experiments  Illustrating 
the  Precautions  to  be  Taken — Experiments  in  Skin-disinfection. 


DETERMINATION   OF   DISINFECTANT   PROPERTIES. 

THERE  are  several  ways  of  determining  the  germicidal 
value  of  chemical  substances,  the  most  common  being  to 
expose  organisms  dried  upon  bits  of  silk  thread  to  the 
disinfectant  for  different  lengths  of  time,  and  then,  after 
removing,  and  carefully  washing  the  threads  in  water,  to 
place  them  in  nutrient  media  at  a  favorable  temperature, 
and  notice  if  any  growth  appears.  If  no  growth  results, 
the  disinfection  is  presumably  successful.  Another  method 
is  to  mix  fluid  cultures  of  bacteria  with  the  disinfectant  in 
varying  proportions,  and,  after  different  intervals  of  time, 
to  determine  if  disinfection  is  in  progress  by  transferring  a 
portion  of  the  mixture  to  nutrient  media,  just  as  in  the  other 
methods  of  work. 

By  the  first  of  these  processes  the  bits  of  thread,  usually 
about  1  to  2  cm.  long,  are  placed  in  a  dry  test-tube  provided 
with  a  cotton  plug  and  carefully  sterilized,  either  by  the 
dry  method  or  in  the  steam  sterilizer,  before  using.  They 
are  then  immersed  in  a  pure  bouillon  culture  or  in  a  salt- 
solution  suspension  of  the  organism  upon  which  the  disin- 
fectant is  to  be  tested.  I  say  "pure  culture,"  because  it  is 
always  desirable  in  testing  a  substance  to  determine  its 
germicidal  value  for  several  different  resistant  species  of 

(331) 


332     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

bacteria,  both  in  the  vegetating  and  in  the  spore  stage,  and 
also  because  it  is  only  by  the  use  of  pure  cultures  of  familiar 
species  that  it  is  possible  to  distinguish  between  the  colonies 
growing  from  the  individuals  that  have  not  been  destroyed 
by  the  disinfectant  under  investigation  and  those  of  unknown 
species  that  may  appear  upon  the  plate  as  contaminations 
occurring  during  the  manipulation. 

After  the  threads  have  remained  in  the  culture  or  suspen- 
sion for  from  five  to  ten  minutes  they  are  removed  under 
aseptic  precautions  and  carefully  separated  and  spread 
upon  the  bottom  of  a  sterilized  Petri  dish,  which  is  then 
placed  either  in  the  incubator  at  a  temperature  not  exceed- 
ing 38°  C.  until  the  excess  of  fluid  has  evaporated,  or  in  a 
desiccator  over  sulphuric  acid,  calcium  chloride,  or  any 
other  drying-agent.  The  threads  are  not  left  there  until 
absolutely  dry,  but  only  until  the  excess  of  moisture  has 
evaporated.  When  sufficiently  dry  they  are  immersed  in 
solutions  of  the  disinfectant  of  different  but  known  strengths 
for  a  fixed  interval  of  time,  say  one  or  two  hours,  after  which 
they  are  removed,  rinsed  in  sterilized  distilled  water  to 
remove  the  excess  of  disinfectant  adhering  to  them,  and 
placed  in  fresh,  sterile  culture-media,  which  are  then  placed 
in  the  incubator  at  from  37°  to  38°  C.  If  after  twenty-four 
to  forty-eight  or  seventy-two  hours  a  growth  occurs  at  or 
about  the  bit  of  thread,  and  if  this  growth  consists  of  the 
organism  with  which  the  test  was  made,  manifestly  there 
has  been  no  disinfection;  if  no  growth  occurs  after,  at  most, 
ninety-six  hours,  it  is  safe  to  presume  that  the  bacteria 
have  been  killed,  unless  our  efforts  at  rinsing  off  the  excess  of 
disinfectant  from  the  thread  have  not  been  successful,  and 
a  small  amount  of  disinfectant  is  still  active  in  preventing 
development — i.  e.,  is  acting  as  an  antiseptic. 


DETERMINATION  OF  DISINFECTANT  PROPERTIES    333 

By  the  process  in  which  cultures  or  suspensions  of  the 
organisms  are  mixed  with  different  but  known  strengths  of 
the  disinfectant  a  small  portion  of  the  mixture,  usually  a 
loopful  or  a  drop,  is  transferred  at  the  end  of  a  definite  time 
to  the  fresh  medium  which  is  to  determine  whether  the 
organisms  have  been  killed  or  not.  This  is  commonly  a 
tube  of  fluid  agar-agar,  which  is  poured  into  a  Petri  dish, 
allowed  to  solidify,  and  placed  in  the  incubator,  as  in  the 
'preceding  method. 

After  the  minimum  strength  of  disinfectant  necessary  to 
destroy  the  vitality  of  the  organisms  with  which  we  are 
Working  has  been  determined  for  any  fixed  time,  it  remains 
for  us  to  decide  what  is  the  shortest  time  in  which  this  strength 
will  have  the  same  effect.  We  then  work  with  a  constant 
dilution  .of  the  disinfectant,  but  with  varying  intervals  of 
exposure — one,  five,  •  ten  minutes,  etc. — until  we  have 
decided  not  only  the  minimum  amount  of  disinfectant 
required  for  the  destruction  of  the  bacteria,  but  the  shortest 
time  necessary  for  this  under  known  conditions. 

A  factor  not  to  be  lost  sight  of  is  the  temperature  at 
which  these  experiments  are  conducted,  for  it  must  always 
be  borne  in  mind  that  the  action  of  a  disinfectant  is  usually 
more  energetic  at  a  higher  than  at  a  lower  temperature. 

Now  in  both  of  these  methods  it  is  easy  to  see  that  unless 
special  precautions  are  taken  a  minute  portion  of  the  disin- 
fectant may  be  carried  along  with  the  thread,  or  drop,  into 
the  medium  which  is  to  determine  whether  the  organisms 
do  or  do  not  possess  the  power  of  growth,  and  there  have 
a  restraining  or  antiseptic  action.  For  organisms  in  their 
normal  condition — that  is,  those  which  have  never  been 
exposed  to  the  action  of  a  disinfectant — the  amount  of 
certain  disinfectants  that  is  necessary  to  restrain  growth 


334     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

is  very  small  indeed;  but  for  organisms  that  have  already 
been  exposed  for  a  time  to  such  agents  this  amount  is  very 
much  less.  It  is  plain,  then,  that  if  the  test  is  to  be  an 
accurate  one,  precautions  must  be  taken  against  admitting 
this  minute  trace  of  disinfectant  to  the  medium  with  which 
we  are  to  determine  whether  the  bacteria  exposed  to  the 
disinfectant  were  killed  or  not.  The  precautions  hitherto 
taken  to  prevent  this  accident  have  been,  when  the  threads 
were  employed,  washing  them  in  sterilized  distilled  water 
and  then  in  alcohol;  or,  where  fluid  cultures  were  mixed 
with  the  disinfectant  in  solution,  an  effort  was  usually 
made  to  dilute  the  amount  of  disinfectant  carried  over,  to 
a  point  at  which  it  lost  its  inhibiting  power. 

While  such  precautions  are  sufficient  in  many  cases, 
they  do  not  answer  for  all.  Certain  chemicals  have  the 
property  of  combining  so  firmly  with  the  threads  upon 
which  the  bacteria  are  located  as  to  require  other  special 
means  of  ridding  the  threads  of  them;  and  in  solutions  in 
which  proteid  substances  are  present  along  with  the  bacteria 
a  similar  union  between  them  and  the  disinfectant  may 
likewise  take  place.  In  both  instances  this  amount  of  disin- 
fectant adhering  to  the  threads  or  in  combination  with  the 
proteids  must  be  eliminated,  otherwise  the  results  of  the 
test  may  be  fallacious.  A  partial  solution  of  the  problem 
is  given  through  studies  that  have  been  made  upon  corrosive 
sublimate  in  its  various  applications  for  disinfecting  pur- 
poses, and  in  this  connection  it  has  been  shown  by  Shaefer1 
that  it  is  impossible  to  rid  silk  threads  of  the  corrosive 
sublimate  adhering  to  them  by  simple  washing,  as  the  sub- 
limate acts  as  a  mordant  and  forms  a  firm  union  with  the 

1  Berliner  klin.  Wochenschrift,  1890,  No.  3,  p.  50. 


DETERMINATION  OF  DISINFECTANT  PROPERTIES    335 

tissues  of  the  threads.  Braatz1  found  the  same  to  hold  good 
for  catgut.  For  example,  he  found  that  catgut  which  had 
been  immersed  in  solutions  of  corrosive  sublimate  gave  the 
characteristic  reactions  of  the  salt  after  having  been  im- 
mersed for  five  weeks  in  distilled  water  which  had  been 
repeatedly  renewed.  Braatz  remarks  that  a  similar  com- 
bination between  sublimate  and  cotton  will  take  place  after 
a  long  time;  but  it  occurs  so  slowly  that  it  cannot  interfere 
with  disinfection  experiments  in  the  same  way  that  silk  does. 

The  most  successful  attempt  at  removing  all  traces  of 
sublimate  from  the  threads  or  from  the  proteid  substances 
in  which  are  located  the  bacteria  whose  vitality  is  to  be 
tested  was  made  by  Geppert,  who  subjected  them  to  the 
action  of  ammonium  sulphide  in  solution.  By  this  procedure 
the  mercury  is  converted  into  inert,  insoluble  sulphide,  and 
has  no  inhibiting  effect  upon  the  growth  of  those  bacteria 
that  did  not  succumb  to  its  action  when  in  the  form  of  the 
bichloride. 

Another  plan  that  has  been  successfully  used  is  to  dry 
the  bacteria  on  small  particles  of  sterile  glass  rod  or  on 
sterile  glass  beads  instead  of  on  threads.  The  advantages 
of  the  method  are  obvious,  but  the  handling,  especially  the 
washing,  must  be  done  carefully  or  all  the  bacteria  will  be 
removed  from  the  glass  surfaces. 

In  the  second  method  of  testing  disinfectants  mentioned 
above — that  is,  when  cultures  of  bacteria  and  solutions 
of  the  disinfectant  are  mixed,  and  after  a  time  a  drop  of  the 
mixture  is  removed  and  added  to  sterile  nutrient  media — 
the  inhibiting  amount  of  disinfectant  can  readily  be  got 
rid  of  by  dilution;  that  is  to  say,  instead  of  transferring  the 

1  Centralblatt  fur  Bakteriologie  und  Parasitenkunde,  Bd.  vii,  No.  1,  p.  8. 


336     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

drop  directly  to  the  fresh  medium,  add  it  to  10  or  12  c.c. 
of  sterilized  salt-solution  (0.6-0.7  per  cent,  of  NaCl  in  dis- 
tilled water)  or  distilled  water,  and  after  thoroughly  shaking 
add  a  drop  of  this  to  the  medium  in  which  the  power  of 
development  of  the  bacteria  is  to  be  determined. 

Another  important  point  to  be  borne  in  mind  in  testing 
disinfectants  is  the  necessity  of  so  adjusting  the  conditions 
that  each  individual  organism  will  be  exposed  to  the  action 
of  the  agent  used.  When  clumps  of  bacteria  exist  we  are 
not  always  assured  of  this,  for  only  those  on  the  surface 
of  the  clump  may  be  affected,  while  those  in  the  center  of 
the  mass  may  escape,  being  protected  by  those  surrounding 
them.  These  clumps  and  minute  masses  are  especially 
liable  to  be  present  in  fluid  cultures  and  in  suspensions  of 
bacteria,  and  must  be  eliminated  before  the  test  is  begun, 
if  this  is  to  be  made  by  mixing  them  with  solutions  of  the 
agent  to  be  tested.  This  is  best  accomplished  in  the  following 
way:  the  organisms  should  be  cultivated  in  bouillon  con- 
taining sand  or  finely  divided  particles  of  glass;  after  grow- 
ing for  a  sufficient  length  of  time  they  are  to  be  shaken 
thoroughly,  in  order  that  all  clumps  may  be  mechanically 
broken  up  by  the  sand.  The  culture  is  then  filtered  through 
a  tube  containing  closely  packed  glass-wool. 

The  filtration  may  be  accomplished  without  fear  of  con- 
tamination of  the  culture  by  the  employment  of  an  Allihin 
tube,  which  is  practically  a  thick-walled  test-tube  drawn  out 
to  a  finer  tube  at  its  blunt  end  so  as  to  convert  it  into  a 
sort  of  cylindrical  funnel.  The  tube  when  ready  for  use 
has  the  appearance  shown  in  Fig.  65. 

This  tube,  after  being  plugged  at  the  bottom  with  glass- 
wool  (a,  Fig.  65),  and  at  its  wide  extremity  with  cotton- 
wool, is  placed  vertically,  small  end  down,  into  an  Erlen- 


DETERMINATION  OF  DISINFECTANT  PROPERTIES    337 


FIG.  65 


meyer  flask  of  about  100  c.c.  capacity  and  sterilized  in  a 
steam  sterilizer  for  the  proper  time.  It  is  kept  in  the  steril- 
izer until  it  is  to  be  used,  which  should 
be  as  soon  as  possible  after  sterilization. 

The  watery  suspension  or  bouillon  cul- 
ture of  the  organisms  is  now  to  be  filtered 
repeatedly  through  the  glass-wool  into 
sterilized  flasks  until  a  degree  of  trans- 
parency is  reached  which  will  permit  the 
reading  of  moderately  fine  print  through 
a  layer  of  the  fluid  about  2  cm.  thick 
— i.  e.,  through  an  ordinary  test-tube 
full  of  it.  This  filtrate  can  then  be  sub- 
jected to  the  action  of  the  disinfectant. 
As  a  rule,  the  results  are  more  uniform 
than  when  no  attention  is  paid  to  the 
presence  of  clumps.  It  is  scarcely  neces- 
sary to  say  that  in  the  practical  employ- 
ment of  disinfectants  outside  the  labora- 
tory no  such  precautions  are  taken;  but 
in  laboratory,  work,  where  it  is  desired 
to  determine  exactly  the  value  of  different 
substances  as  germicides,  all  the  precau- 
tions mentioned  will  be  found  essential 
to  precision. 

The  disinfectant  value  of  gases  and 
vapors  is  determined  by  their  action  Cylindrical  funnel 

used  for  filtering  cul- 

upon  test-objects  in  closed  chambers,  tures  on  which  dis- 
The  object  is  to  determine  the  proportion  ^fsetfdants  are  to  be 
of  the  gas,  when  mixed  with  air,  that  is 
required  to  destroy  the  bacteria  exposed  to  its  action  in 
a  given  time.  For  this  purpose  the  test  is  usually  made 
22 


IP* 


338     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

as  follows:  under  a  sterilized  bell-glass  of  known  capacity 
the  test-objects  are  placed.  Into  the  chamber  is  then 
admitted  sufficient  of  a  mixture  of  air  and  the  gas  under 
consideration,  of  known  proportions,  to  displace  com- 
pletely all  the  air;  or  the  pure  gas  itself  may  be  intro- 
duced in  amount  necessary  to  give  the  desired  dilution 
when  mixed  with  the  air  in  the  chamber.  At  the  expiration 
of  the  time  decided  upon  for  the  test  the  infected  articles 
are  removed  and  the  vitality  of  the  bacteria  upon  them  is 
determined. 

In  the  case  of  vapors  of  volatile  fluids,  such,  for  instance, 
as  formalin,  the  fluid  is  placed  under  the  bell-glass  in  an 
open  dish;  in  another  open  dish  the  test-objects  are  placed. 
The  bell-glass  is  then  sealed  to  an  underlying  ground-glass 
plate  by  vaseline  or  paraffin,  and  the  fluid  is  allowed  to 
vaporize  at  ordinary  room-temperature.  The  point  here 
to  be  decided  is  the  volume  or  weight  of  such  a  fluid  that 
it  is  necessary  to  expose  in  an  air-chamber  of  known  cubic 
capacity  in  order  that  bacteria  may  be  destroyed  by  its 
vapor  in  a  given  time. 

In  determining  the  germicidal  value  of  different  chemical 
agents  for  certain  pathogenic  bacteria  susceptible  animals 
are  sometimes  inoculated  with  the  organisms  after  they  have 
been  exposed  to  the  disinfectant.  If  no  pathological  con- 
dition, results,  disinfection  is  assumed  to  have  been  suc- 
cessful; while  if  the  condition  characteristic  of  the  activities 
of  the  given  organism  in  the  tissues  of  this  animal  appears, 
the  reverse  is  the  case.  The  objections  to  this  method  are: 
"First.  The  test-organisms  may  be  modified  as  regards 
reproductive  activity  without  being  killed;  and  in  this 
case  a  modified  form  of  the  disease  may  result  from  the 
inoculation,  of  so  mild  a  character  as  to  escape  observation. 


DETERMINATION  OF  ANTISEPTIC  PROPERTIES     339 

Second.  An  animal  that  has  suffered  this  modified  form  of 
the  disease  enjoys  protection,  more  or  less  perfect,  from 
future  attacks,  and  if  used  for  a  subsequent  experiment  may, 
by  its  immunity  from  the  effects  of  the  pathogenic  test- 
organism,  give  rise  to  the  mistaken  assumption  that  this 
had  been  destroyed  by  the  action  of  the  germicidal  agent 
to  which  it  had  been  subjected."  (Sternberg.) 

DETERMINATION   OF   ANTISEPTIC   PROPERTIES. 

For  this  purpose  sterile  media  are  employed,  and  are 
usually  arranged  in  two  groups:  the  one  to  remain  normal 
in  composition  and  to  serve  as  controls,  while  to  the  other  the 
substance  to  be  tested  is  to  be  added  in  different  but  known 
strengths.  It  is  customary  to  employ  test-tubes  each  con- 
taining an  exact  amount  of  bouillon,  gelatin,  or  agar-agar, 
as  the  case  may  be.  To  each  tube  a  definite  amount  of  the 
antiseptic  is  added,  and  if  it  is  not  of  a  volatile  nature  or 
not  injured  by  heat,  the  tubes  may  then  be  sterilized. 
After  this  they  are  to  be  inoculated  with  the  organism 
with  which  the  test  is  to  be  made,  and  at  the  same  time  one 
of  the  "  control' '-tubes  (one  of  those  to  which  no  antiseptic 
has  been  added)  is  inoculated.  They  are  all  then  to  be 
placed  in  the  incubator  and  kept  under  observation.  If 
at  the  end  of  twenty-four,  forty-eight,  or  seventy-two  hours 
no  growth  appears  in  any  but  the  "control' '-tubes,  it  is 
evident  that  the  antiseptic  must  be  added  in  smaller 
amounts,  for  we  are  to  determine  the  point  at  which  it  is 
not  as  well  as  that  at  which  it  is  capable  of  preventing 
development'.  The  experiment  is.  then  repeated,  using 
smaller  amounts  of  the  antiseptic  until  we  reach  a  point  at 
which  growth  just  occurs,  notwithstanding  the  presence 


340     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

of  the  antiseptic;  the  amount  necessary  for  antisepsis  is 
then  a  trifle  greater  than  that  used  in  the  last  tube.  If, 
for  example,  there  was  no  development  in  the  tubes  in  which 
the  antiseptic  was  present  in  the  proportion  of  1 : 1000,  and 
growth  in  the  one  in  which  it  was  present  in  1 : 1400,  the 
experiment  should  be  repeated  with  strengths  of  the  anti- 
septic corresponding  to  1:1000,  1:1100,  1:1200,  1:1300, 
1  :  1400,  and  in  this  way  one  ultimately  determines  the 
amount  by  which  growth  is  just  prevented;  this  represents 
the  antiseptic  value  of  the  substance  for  the  organism  with 
which  it  was  tested. 

.  i  EXPERIMENTS. 

To  each  of  three  tubes  containing  10  c.c. — one  of  physio- 
logical salt-solution,  another  of  bouillon,  a  third  of  fluid 
blood  serum — add  as  much  of  a  culture  of  micrococcus 
aureus  as  can  be  held  upon  a  looped  platinum  wire.  Break 
this  up  carefully  to  eliminate  clumps,  and  then  add  exactly 
10  c.c.  of  a  1 : 500  solution  of  corrosive  sublimate.  Mix 
thoroughly,  and  at  the  end  of  three  minutes  transfer  a  drop 
from  each  tube  into  tubes  of  liquefied  agar-agar,  and  pour 
these  into  Petri  dishes.  Label  each  dish  carefully  and  place 
them  in  the  incubator.  Are  the  results  the  same  in  all  the 
plates?  How  are  the  differences  to  be  explained?  To 
what  strength  of  the  disinfectant  were  the  organisms  ex- 
posed in  the  experiment? 

To  each  of  two  tubes — the  one  containing  10  c.c.  of 
physiological  salt-solution,  the  other  of  bouillon— add  as 
much  of  a  spore-containing  culture  of  anthrax  bacilli  as  can 
be  held  upon  a  loop  of  platinum  wire.  Distribute  this  uni- 
formly through  the  medium,  and  then  add  exactly  10  c.c.  of 
a  1 :  500  solution  of  corrosive  sublimate.  Mix  thoroughly, 


EXPERIMENTS  341 

and  at  the  end  of  five  minutes  transfer  a  drop  from  each  tube 
to  tubes  of  liquefied  agar-agar.  Pour  these  immediately  into 
Petri  dishes.  Label  each  dish  carefully  and  place  them  in 
the  incubator.  Note  the  results  at  the  end  of  twenty-four, 
forty-eight,  and  seventy-two  hours.  How  do  you  explain 
them? 

Make  identically  the  same  experiment  with  the  same  spore- 
containing  culture  of  anthrax  bacilli,  except  that  the  drop 
from  the  mixture  is  to  be  transferred  to  10  c.c.  of  a  mixture 
of  equal  parts  of  ammonium  sulphide  and  sterilized  distilled 
water.  After  remaining  in  this  for  about  half  a  minute, 
a  drop  is  to  be  transferred  to  a  tube  of  liquefied  agar-agar, 
poured  into  Petri  dishes,  labelled,  and  placed  in  the  incubator. 
Note  the  results.  Do  they  correspond  with  those  obtained 
in  the  preceding  experiment?  How  are  the  differences 
explained? 

Prepare  a  1 : 1000  solution  of  corrosive  sublimate.  To 
each  of  twelve  tubes  containing  exactly  10  c.c.  of  bouillon, 
add  one  drop  to  the  first,  two  drops  to  the  second,  and  so 
on  until  the  last  tube  has  had  twelve  drops  added  to  it. 
Mix  thoroughly  and  then  inoculate  each  with  one  wire- 
loopful  of  a  bouillon  culture  of  micrococcus  aureus.  Place 
them  all  in  the  incubator  after  carefully  labelling  them. 
Note  the  order  in  which  growth  appears. 

Do  the  same  with  anthrax  spores,  with  spores  of  bacillus 
subtilis,  and  with  the  typhoid  bacillus,  and  compare  the 
results.  From  these  experiments,  what  will  be  the  strength 
of  corrosive  sublimate  necessary  to  antisepsis  under  these 
conditions  for  the  organisms  employed? 

Make  a  similar  series  of  experiments  using  a  5  per  cent, 
solution  of  carbolic  acid. 


342     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

Determine  the  antiseptic  value  of  the  common  disinfec- 
tants for  the  organisms  with  which  you  are  working. 

Determine  the  time  necessary  for  the  destruction  of  the 
organisms  with  which  you  are  working,  by  corrosive  sub- 
limate in  1 : 1000  solution,  under  different  conditions— with 
and  without  the  presence  of  albuminous  bodies  other  than 
the  bacteria,  and  under  varying  conditions  of  temperature. 

In  making  these  experiments  be  careful  to  guard  against 
the  introduction  of  sufficient  sublimate  into  the  agar-agar 
with  which  the  Petri  plate  is  to  be  made  to  inhibit  the  growth 
of  the  organisms  which  may  not  have  been  destroyed  by 
the  sublimate.  This  may  be  done  by  transferring  two  drops 
from  the  mixture  of  sublimate  and  organism  into  not  less 
than  10  c.c.  of  sterilized  physiological  salt  solution,  in  which 
they  may  be  thoroughly  shaken  for  from  one  to  two  minutes, 
or  into  the  solution  of  ammonium  sulphide  of  the  strength 
given. 

To  10  c.c.  of  a  bouillon  culture  of  micrococcw  aureus  or 
anthrax  spores  add  10  c.c.  of  a  1 : 500  solution  of  corrosive 
sublimate,  and  allow  it  to  remain  in  contact  with  the 
organisms  for  only  one-half  the  time  necessary  to  destroy 
them  (use  an  organism  for  which  this  has  been  determined). 
Then  transfer  a  drop  of  the  mixture  to  each  of  three  liquefied 
agar-agar  tubes  and  pour  them  into  Petri  dishes.  Place 
them  in  the  incubator  and  observe  them  for  twenty-four, 
forty-eight,  and  seventy-two  hours.  No  growth  occurs. 
How  is  this  to  be  accounted  for? 

At  the  end  of  seventy-two  hours  inoculate  all  of  these 
plates  with  a  culture  of  the  same  organism  which  has  not 
been  exposed  to  sublimate,  by  taking  up  bits  of  culture  on 


SKIN  DISINFECTION  343 

a  needle  and  drawing  it  across  the  plates.  A  growth  now 
results.  We  have  here  an  experiment  in  which  organisms 
which  have  been  exposed  to  sublimate  for  a  much  shorter 
time  than  necessary  to  destroy  them,  when  transferred 
directly  to  a  favorable  culture  medium  do  not  grow,  and 
yet,  when  the  same  organism  which  has  not  been  exposed 
to  sublimate  at  all  is  planted  upon  the  same  medium  it 
does  grow.  How  is  this  to  be  accounted  for? 

SKIN  DISINFECTION. 

With  a  sterilized  knife  scrape  from  the  skin  of  the  hands, 
at  the  root  of  the  nails,  and  under  the  nails,  small  particles 
of  epidermis.  Prepare  plates  from  them.  Note  the  results. 

Wash  the  hands  carefully  for  ten  minutes  in  hot  water  and 
scrub  them  during  this  time  with  soap  and  a  sterilized 
brush.  Rinse  them  in  hot  water.  Again  prepare  plates 
from  scrapings  of  the  skin  on  the  fingers,  at  the  root  of  the 
nails,  and  under  the  nails.  Note  the  results. 

Again  wash  as  before  in  hot  water  with  soap  and  brush, 
rinse  in  hot  water,  then  soak  the  hands  for  five  minutes  in 
1  :  1000  corrosive  sublimate  solution,  and,  as  before,  prepare 
plates  from  scrapings  from  the  same  localities.  Note  the 
results. 

Repeat  this  latter  procedure  in  exactly  the  same  way, 
but  before  taking  the  scrapings  let  some  one  pour  ammonium 
sulphide  over  the  points  from  which  the  scrapings  are  to 
be  made.  After  it  has  been  on  the  hands  about  three  minutes 
again  scrape,  and  note  the  result  upon  plates  made  from  the 
scrapings. 

Wash  as  before  in  hot  water  and  soap,  rinse  in  clean  hot 
water,  immerse  for  a  minute  or  two  in  alcohol,  after  this  in 


344     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

1  : 1000  sublimate  solution,  and  finally  in  ammonium  sul- 
phide, and  then  prepare  plates  from  scrapings  from  the 
points  mentioned. 

In  what  way  do  the  results  of  these  experiments  differ 
from  one  another? 

To  what  are  these  differences  due? 

What  have  these  experiments  taught? 

In  making  the  above  experiments  it  must  be  remembered 
that  the  strictest  care  is  necessary  in  order  to  prevent  the 
access  of  germs  from  without  into  our  media.  The  hand 
upon  which  the  experiment  is  being  performed  must  be 
held  away  from  the  body  and  must  not  touch  any  object 
not  concerned  in  the  experiment.  The  scraping  should  be 
done  with  the  point  of  a  knife  that  has  been  sterilized  in 
a  flame  and  allowed  to  cool.  The  scrapings  may  be  trans- 
ferred directly  from  the  knife-point  to  the  gelatin  by  means 
of  a  sterilized  platinum  wire  loop. 

The  brush  used  should  be  thoroughly  cleansed  and  always 
kept  in  1  :  1000  solution  of  corrosive  sublimate.  It  should 
-be  washed  in  hot  water  before  using. 


CHAPTER  XIX. 

Micrococcus  Aureus — Micrococcus  Pyogenes  and  Citreus — Staphylococcus 
Epidermidis  Albus — Streptococcus  Pyogenes — Micrococcus  Gonor- 
rhcese — Micrococcus  Intracellularis — Pseudomonas  ^Eruginosa — Bacil- 
lus of  Bubonic  Plague. 

MICROCOCCUS  AUREUS  (ROSENBACH),  MIGULA,  1900. 

SYNONYMS:  Staphylococcus  pyogenes  aureus,  Rosenbach,  1884;  Micro- 
coccus  pyogenes  aureus,  Migula,  1895;  Micrococcus  pyogenes,  Lehmann  and 
Neumann,  1896. 

PREPARE  a  set  of  plates  of  agar-agar  from  the  pus  of  an 
acute  abscess  or  boil  that  has  been  opened  under  antiseptic 
precautions.  Care  must  be  taken  that  none  of  the  antiseptic 
used  gains  access  to  the  culture  tubes,  otherwise  its  restrain- 
ing effect  may  be  operative  and  the  development  of  the 
organisms  interfered  with.  It  is  best,  therefore,  to  take  a 
drop  of  the  pus  upon  a  platinum-wire  loop  after  it  has  been 
flowing  for  a  few  seconds;  even  then  it  must  be  taken 
from  the  mouth  of  the  incision  and  before  it  has  run  over 
the  surface  of  the  skin.  At  the  same  time  prepare  two  or 
three  coverslips  from  the  pus. 

Microscopic  examination  of  these  slips  will  reveal  the 
presence  of  a  large  number  of  pus-cells,  both  multinucleated 
and  with  horseshoe-shaped  nuclei,  some  threads  of  disin- 
tegrated and  necrotic  connective  tissue,  and,  lying  here 
and  there  throughout  the  preparation,  small  round  bodies 
which  will  sometimes  appear  singly,  sometimes  in  pairs, 
and  frequently  will  be  seen  grouped  together  somewhat  like 
clusters  of  grapes.  (See  Fig.  66.)  They  stain  readily  and 

(345) 


346     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

are  commonly  located  in  the  material  between  the  pus-cells; 
very  rarely  they  may  be  seen  in  the  protoplasmic  body  of 
the  cell.  (Compare  the  preparation  with  a  similar  one  made 
from  the  pus  of  gonorrhea.  (See  Fig.  69.)  In  what  way  do 
the  two  preparations  differ,  the  one  from  the  other? 

After  twenty-four  hours  in  the  incubator  the  plates  will 
be  seen  to'  be  studded  here  and  there  with  yellow  or  orange- 
colored  colonies,  which  are  usually  round,  moist,  and  glis- 
tening in  their  naked-eye  appearances.     When  located  in 
.  .  \ 

FIG.  66 


/  I  A   f 


o 


Preparation  from  pus,  showing  pus-cells,  A,  and  micrococci,  C. 

the  depths  of  the  medium  they  are  commonly  seen  to  be 
lozenge  or  whetstone  in  shape,  while  often  they  appear  as 
irregular  stars  with  blunt  points,  and  again  as  irregularly 
lobulated  dense  masses.  In  structure  they  are  conspicuous 
for  their  density.  Under  the  low  objective  they  appear, 
when  on  the  surface,  as  coarsely  granular,  irregularly  round 
patches,  with  more  or  less  ragged  borders  and  a  dark  irreg- 
ular central  mass,  which  has  somewhat  the  appearance  of 
masses  of  coarser  clumps  of  the  same  material  as  that  com- 


MICROCOCCUS  AUREUS  347 

posing  the  rest  of  the  colony.  Microscopically,  these  colo- 
nies are  composed  of  small  round  cells,  irregularly  grouped 
together.  They  are  in  every  way  of  the  same  appearance  as 
those  seen  upon  the  original  cover-slip  preparation. 

Prepare  from  one  of  these  colonies  a  pure  stab-culture  in 
gelatin.  After  thirty-six  to  forty-eight  hours  liquefaction 
of  the  gelatin  along  the  track  of  the  needle,  most  conspicu- 
ous at  its  upper  end,  will  be  observed.  As  growth  continues 
the  liquefied  portion  becomes  more  or  less  of  a  stocking- 
shape,  and  gradually  widens  at  its  upper  end  into  an  irregular 
funnel.  This  will  continue  until  the  whole  of  the  gelatin 
in  the  tube  eventually  becomes  fluid.  There  can  always  be 
noticed  at  the  bottom  of  the  liquefying  portion  an  orange- 
colored  or  yellow  mass  composed  of  a  number  of  the  organ- 
isms which  have  sunk  to  the  bottom  of  the  fluid. 

On  potato  the  growth  is  quite  luxuriant,  appearing  as  a 
brilliant,  orange-colored  layer,  somewhat  lobulated  and  a 
little  less  moist  than  when  growing  upon  agar-agar. 

It  does  not  produce  fermentation  with  gas  production. 

It  belongs  to  the  group  of  facultative  anaerobes. 

In  .milk  it  causes  coagulation  with  acid  reaction.  This 
is,  however,  variable. 

It  is  not  motile,  and  being  of  the  family  of  micrococci 
does  not  form  endogenous  spores.  It  possesses,  however, 
a  degree  of  resistance  to  detrimental  agencies  that  is  some- 
what greater  than  that  common  to  non-spore-bearing 
bacteria. 

In  bouillon  it  causes  a  diffuse  clouding,  and  after  a  time 
a  yellow  or  orange-colored  sedimentation. 

This  organism  is  the  commonest  of  the  pathogenic  bacteria 
with  which  we  shall  meet.  It  is  micrococcus  aureus,  or  as 
it  is  more  commonly  known,  the  staphylococcus  aureus, 


348     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

and  is  the  organism  most  frequently  concerned  in  the  pro- 
duction of  acute,  circumscribed,  suppurative  inflammations. 
As  it  is  almost  ubiquitous,  it  is  a  source  of  continuous 
annoyance  to  the  surgeon. 

While  it  is  the  etiological  factor  in  the  production  of  most 
of  the  suppurative  processes  in  man,  still  it  is  with  no  little 
difficulty  that  these  conditions  can  be  reproduced  experi- 
mentally in  lower  animals.  Its  subcutaneous  introduction 
into  their  tissues  does  not  always  result  in  abscess  formation, 
and  when  it  does  there  is  probably  coincident  interference 
with  the  circulation  and  nutrition  of  these  tissues  which 
renders  them  less  able  to  resist  its  inroads.  When  intro- 
duced into  the  great  serous  cavities  of  the  lower  animals 
its  presence  is  likewise  not  always  accompanied  by  the 
production  of  inflammation.  If  the  abdominal  cavity  of 
a  dog,  for  example,  be  carefully  opened  so  as  to  make  as 
slight  a  wound  as  possible,  and  no  injury  be  done  to  the 
intestines,  large  quantities  of  bouillon  cultures  or  watery 
suspensions  of  this  organism  may  be,  and  repeatedly  have 
been  introduced  into  the  peritoneum  without  the  slightest 
injury  to  the  animal.  On  the  contrary,  if  some  substance 
which  acts  as  a  direct  irritant  to  the  intestines — such,  for 
example,  as  a  small  bit  of  potato  upon  which  the  organisms 
are  growing— be  at  the  same  time  introduced,  or  the  intes- 
tines be  mechanically  injured,  so  that  there  is  a  disturbance 
in  their  circulation,  then  the  introduction  of  these  organisms 
is  promptly  followed  by  acute  and  fatal  peritonitis.  (Hal- 
sted.1) 

On  the  other  hand,  the  results  which  follow  their  introduc- 
tion into  the  circulation  are  practically  constant.  If  one 

1  The  Johns  Hopkins  Hospital  Reports.  Report  in  Surgery,  No.  1, 
1891,  ii,  No.  5,  301-303. 


MICROCOCCUS  A  U  RE  US  349 

inject  into  the  circulation  of  the  rabbit  through  a  vein  of 
the  ear,  or  in  any  other  way,  from  0.1  to  0.3  c.c.  of  a  bouillon 
culture  or  watery  suspension  of  a  virulent  variety  of  this 
organism,  a  fatal  pyemia  always  follows  in  from  two  and 
one-half  to  three  days.  A  few  hours  before  death  the  animal 
suffers  frequently  from  severe  convulsions.  Now  and  then 
excessive  secretion  of  urine  is  noticed.  The  animal  may 
appear  in  moderately  good  condition  until  from  eight  to 
ten  hours  before  death.  At  the  autopsy  a  typical  picture 
presents:  the  voluntary  muscles  are  seen  to  be  marked 
here  and  there  by  yellow  spots,  which  average  the  size  of  a 
flaxseed,  and  are  of  about  the  same  shape.  They  lie  usu- 
ally with  their  long  axis  running  parallel  to  the  muscle- 
fibers.  As  the  abdominal  and  thoracic  cavities  are  opened 
the  diaphragm  is  often  seen  to  be  studded  with  them. 
Frequently  the  pericardial  sac  is  distended  with  a  clear 
gelatinous  fluid,  and  almost  constantly  the  yellow  points 
are  seen  in  the  myocardium.  The  kidneys  are  rarely  with- 
out them ;  here  they  appear  on  the  surface  as  isolated  yellow 
points,  or,  again,  are  seen  as  conglomerate  masses  of  small 
yellow  points  which  occupy,  as  a  rule,  the  area  fed  by  a 
single  vessel.  If  one  make  a  section  into  one  of  these 
yellow  points,  it  will  be  seen  to  extend  deep  down  through 
the  substance  of  the  kidney  as  a  yellow,  wedge-shaped  mass, 
the  base  of  the  wedge  being  at  the  surface  of  the  organ. 

It  is  very  rare  that  these  abscesses — for  abscesses  the 
yellow  points  are,  as  we  shall  see  when  we  come  to  study 
them  more  closely — are  found  either  in  the  liver,  spleen, 
or  brain;  their  usual  location  being,  as  said,  in  the  kidney, 
myocardium,  and  voluntary  muscles. 

These  minute  abscesses  have  a  dry,  cheesy,  necrotic 
center,  in  which  the  micrococci  are  present  in  large  numbers 


350     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

as  may  be  seen  upon  cover-slips  and  in  cultures  prepared 
for  them. 

Preserve  in  alcohol  bits  of  all  tissues  in  which  the  abscesses 
are  located.  When  these  tissues  are  hard  enough  to  cut 
sections  should  be  made  through  the  abscess  points  and  the 
histological  changes  carefully  studied. 

Microscopic  Study  of  Cover-slips  and  Sections. — In  cover- 
slip  preparations  this  organism  stains  readily  with  the 
ordinary  dyes.  In  tissues,  however,  it  is  best  to  employ 
some  method  by  means  of  which  contrast-stains  may  be 
utilized,  and  the  location  and  grouping  of  the  organisms  in 
the  tissues  rendered  more  conspicuous.  When  stained,  sec- 
tions of  tissues  containing  the  small  abscesses  present  the 
following  appearances: 

To  the  naked  eye  will  be  seen  here  and  there  in  the  section, 
if  the  abscesses  are  very  numerous,  small,  darkly  stained 
areas  which  range  in  size  from  that  of  a  pin-point  up  to 
those  having  a  diameter  of  from  1  to  2  mm.  These  points, 
when  in  the  kidney,  may  be  round  or  oval  in  outline;  or 
may  appear  wedge-shaped,  with  the  base  of  the  wedge 
toward  the  surface  of  the  organ.  The  differences  in  shape 
depend  frequently  upon  the  direction  in  which  the  section 
has  been  made  through  the  kidney.  In  the  muscles  they 
are  irregularly  round  or  oval. 

When  quite  small  they  appear,  in  stained  sections,  to  the 
naked  eye,  as  simple,  round  or  oval,  darkly  stained  points; 
but  when  they  are  in  a  more  advanced  stage  a  pale  center 
can  usually  be  made  out. 

.  When  magnified  they  appear  in  the  earliest  stages  as 
minute  aggregations  of  small  cells,  the  nuclei  of  which  stain 
intensely.  Almost  always  evidences  of  progressing  necrosis 
can  be  seen  about  the  center  of  these  cell-accumulations. 


MICROCOCCUS  AUREUS  351 

The  normal  structure  of  the  cells  of  the  tissues  is  more  or 
less  destroyed;  there  is  seen  a  granular  condition  due  to 
cell-fragmentation;  at  different  points  about  the  center 
of  this  area  the  tissue  appears  cloudy  and  the  tissue-cells 
do  not  stain  readily.  Round  about  and  through  this  spot 
are  seen  the  nuclei  of  pus-cells,  many  of  which  are  under- 
going disintegration.  In  the  smallest  of  these  beginning 
abscesses  the  micrococci  are  to  be  seen  scattered  about  the 
center  of  the  necrotic  tissue;  but  in  a  more  advanced  stage 
they  are  commonly  seen  massed  together  in  very  large 
numbers  in  the  form  commonly  referred  to  as  emboli  of 
micrococci,  meaning,  obviously,  that  they  had.  developed 
within  the  lumen  of  a  tiny  bloodvessel. 

When  the  process  is  well  advanced,  the  different  parts 
of  the  abscess  are  more  easily  detected.  They  then  present 
in  sections  somewhat  the  following  conditions:  at  the 
center  can  be  seen  a  dense,  granular  mass  which  stains  readily 
with  the  basic  aniline  dyes,  and  when  highly  magnified  is 
found  to  be  made  up  of  micrococci.  Sometimes  the  shape 
of  this  mass  of  micrococci  corresponds  to  that  of  the  capil- 
lary in  which  the  organisms  became  lodged  and  developed. 
Immediately  about  the  embolus  of  cocci  the  tissues  are  in  an 
advanced  stage  of  necrosis.  Their  structure  is  almost  com- 
pletely destroyed,  although  the  destruction  is  seen  to  be 
more  advanced  in  some  of  the  elements  of  the  tissues  than 
in  others.  As  we  approach  the  periphery  of  this  faintly 
stained  necrotic  area  it  becomes  marked  here  and  there 
with  granular  bodies,  irregular  in  size  and  shape,  which 
stain  in  the  same  way  as  do  the  nuclei  of  the  pus-cells  and 
represent  the  result  of  disintegration  going  on  in  these  cells. 

Beyond  this  area  we  come  upon  a  dense,  deeply  stained 
zone,  consisting  of  closely  packed  pus-cells;  of  granular 


352     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

detritus  resulting  from  destructive  processes  acting  upon 
these  cells;  and  of  the  normal  cellular  and  connective-tissue 
elements  of  the  part.  Here  and  there  through  this  zone 
will  be  seen  localized  areas  of  beginning  death  of  the  tissues. 
This  zone  gradually  fades  away  into  the  healthy  surrounding 
tissues.  It  constitutes  the  so-called  "abscess  wall." 

Such  is  the  picture  presented  by  the  miliary  abscess 
when  produced  experimentally  in  the  rabbit,  and  it  corre- 
sponds from  beginning  to  end  with  the  pathological  changes 
which  accompany  the  formation  of  larger  abscesses  in  the 
tissues  of  human  beings. 

From  these  small  abscesses  in  the  tissues  of  the  rabbit 
micrococcus  aureus  may  again  be  obtained  in  pure  culture, 
and  will  present  identically  the  same  characteristics  that 
were  possessed  by  the  culture  with  which  the  animal  was 
inoculated. 

A  characteristic  of  all  staphylococcus  abscesses,  small  as 
well  as  large,  is  centralized  death  of  tissue;  even  in  those  of 
microscopic  dimensions  this  area  of  necrosis  is  always 
discernible  by  appropriate  methods  of  examination.  It 
represents  the  very  starting-point  of  the  destructive  process, 
and  is  referable  to  the  combined  action  of  the  endotoxins 
of  the  bacteria  and  the  interference  with  the  circulation 
of  the  past  due  to  proliferation  of  cells  about  the  point  at 
which  the  bacteria  are  located. 

As  a  result  of  the  studies  of  van  de  Velde,  Krauss,  von 
Lingelsheim,  Neisser  and  Wechsberg,  and  others,  our  knowl- 
edge of  the  poison  that  causes  the  destruction — staphylptoxin, 
as  it  is  called — has  been  greatly  extended.  Through  the 
work  of  these  investigators  we  now  know  that  the  patho- 
genic properties  of  micrococcus  aureus  are  due  to  a  definite 
soluble  toxin  elaborated  by  it:  that  this  poison  is  produced 


MICROCOCCUS  A  U RE  US  353. 

under  artificial  conditions  of  cultivation,  and  may  be  sepa- 
rated from  the  living  organisms  by  filtration;  that  when 
injected  into  the  living  animal  body  its  effects  upon  the 
tissues  are  essentially  reproductions  of  those  accompanying 
the  growth  of  the  organism  itself;  that  when  this  action 
is  tested  upon  particular  cells,  such  as  erythrocytes  and 
leukocytes,  two  distinct  properties  are  exhibited,  one  a 
hemolytic,  through  which  the  red  corpuscles  are  dissolved, 
the  other  a  leucocidic,  through  which  the  white  blood-cor- 
puscles are  destroyed;  that  the  hemolytic  and  leucocidic 
properties  are  distinct  from  one  another,  and  are  due  to  the 
activities  of  two  lysins,  of  which  the  staphylotoxin  is  (in 
part?)  composed,  and  which  may  be  separated  from  one 
another  by  appropriate  methods  of  analysis;  that  the  result 
of  the  treatment  of  animals  with  gradually  increasing  non- 
fatal  doses  of  staphylotoxin  is  the  appearance  in  the  blood 
of  the  animals  of  antibodies  (antilysins)  that  inhibit  the 
action  of  the  toxins  (lysins) ;  and,  finally,  that  in  the  serum 
of  certain  animals  (man  and  horse)  similar  antilysins  in 
varying  amounts  are  normally  present.1 

Petersen,  Paltchikowsky,  Proscher,  and  others  have 
recently  attempted  to  prepare  an  antistaphylococcus  serum 
with  the  following  results:  The  serum  of  patients  recov- 
ering from  severe  staphylococcus  infections  contains  pro- 
tective substances  which  serve  to  protect  rabbits  from  twice 
the  fatal  dose  of  a  staphylococcus  culture.  Similarly  the 
serum  of  immunized  rabbits  and  goats,  as  shown  by  the 
experiments  of  Petersen,  possesses  about  the  same  degree 

1  See  van  de  Velde,  Annales  de  I'Institut  Pasteur,  tome  x,  p.  580;  Krauss 
Wiener  klin.  Wochenschrift,  1900,  No.  3;  Von  Lingelsheim,  Etiologie  und 
Therapie  der  Staphylokoken  Infektion  (monograph),  Berlin-Wien,  1900; 
Neisser  and  Wechsberg,  Zeitschrift  fur  Hygiene  und  Infektionskrankheiten, 
1901,  Bd.  xxxvi,  S.  299. 
23 


354     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

of  protective  powers.  No  antitoxic  power  could  be  demon- 
strated in  the  serum  of  the  treated  animals.  The  extremely 
limited  degree  of  the  protective  power  of  antistaphylo- 
coccus  serums  makes  them  useless  for  curative  purposes  in 
human  beings,  as  Petersen  calculated  that  an  adult  would 
require  from  350  to  700  c.c.  of  the  serum  at  a  single  dose, 
as  judged  by  its  effects  on  the  lower  animals. 

OTHER  COMMON  PYOGENIC  ORGANISMS. 

MICROCOCCUS  PYOGENES  (Rosenbach),  Migula,  1900.  Synonyms: 
Staphylococcus  pyogenes  albus,  Rosenbach,  1884;  Micrococcus  pyo- 
genes  albus,  Lehmann  and  Neumann,  1896. 

MICROCOCCUS  CITREUS  (Passet),  Migula,  1900.  Synonym:  Staphy- 
lococcus pyogenes  citreus,  Passet,  1895. 

The  pus  of  an  acute  abscess  in  the  human  being  may 
sometimes  contain  organisms  other  than  micrococcus  aureus. 
Micrococcus  pyogenes  and  micrococcus  citreus  may  be  found. 
The  colonies  of  the  former  are  white,  those  of  the  latter 
are  lemon  yellow.  With  these  exceptions  they  are  in  all 
essential  cultural  peculiarities  similar  to  micrococcus  aureus. 
As  a  rule,  they  are  not  virulent  for  animals,  and  when  they 
do  possess  pathogenic  properties,  it  is  in  a  much  lower 
degree  than  is  commonly  the  case  with  the  golden  micro- 
coccus.  Streptococcus  pyogenes  is  also  present  sometimes. 
The  commonest  of  the  pyogenic  organisms,  however,  is 
that  just  described,  viz. :  micrococcus  aureus. 

An  organism  that  is  almost  universally  present  in  the 
skin,  and  is  often  concerned  in  producing  mild  forms  of 
inflammation,  is  Staphylococcus  epidermidis  albus  (Welch), 
an  organism  that  readily  may  be  confused  with  micrococcus 
pyogenes.  It  differs  from  the  latter  by  the  slowness  with 
which  it  liquefies  gelatin  and  by  the  comparative  absence 


STREPTOCOCCUS  PYOGENES  355 

of  pathogenic  properties  when  injected  into  the  circulation 
of  rabbits.  Welsh  regards  this  organism  as  a  variety  of 
micrococcus  pyogenes. 

STREPTOCOCCUS  PYOGENES  (ROSENBACH),  MIGULA, 

1900. 

SYNONYMS:  Streptococcus,  Billroth,  1874;  Streptococcus  pyogenes, 
Rosenbach,  1884. 

From  a  spreading  phlegmonous  inflammation  prepare 
cover-slips  and  cultures.  What  is  the  predominating 
organism?  Does  it  appear  in  the  form  of  irregular  clusters 

FIG.  67 

»• 

UV* 


jgjf«I 

**r»»>  / 

?«£»& 

^  •" 


Streptococcus  pyogenes  in  pus. 

like  those  of  grapes,  or  have  its  individuals  a  definite, 
regular  arrangement?  (See  Fig.  67.)  Are  its  colonies  like 
those  of  micrococcus  aureus? 

Isolate   this  organism  in  pure  cultures.     In  these  cul- 


356     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

tures  it  will  be  found  on  microscopic  examination  to  present 
an  arrangement  somewhat  like  a  chain  of  beads.  (Fig.  68.) 

Its  peculiarities  should  be  as  follows: 

Upon  microscopic  examination  a  micrococcus  should  be 
found,  but  differing  in  its  arrangement  from  those  just 
described.  The  single  cells  are  not  scattered  irregularly  or 
arranged  in  clumps  similar  to  bunches  "of  grapes,  but  are 
joined  together  in  chains  like  strands  of  beads.  These 
strands  are  sometimes  regular  in  the  arrangement  and  size 
of  the  individual  cells  composing  them,  but  more  commonly 
certain  irregular  groups  may  be  seen  in  them,  appearing 

FIG.  68 


/,/ 


Streptococcus  pyogenes. 

as  if  two  or  three  cells  had  fused  together  to  form  a  link 
in  the  chain,  so  to  speak,  that  is  somewhat  longer  than  the 
others;  again,  portions  of  the  chain  may  be  thinner  than 
the  rest,  or  it  may  appear  broken  or  ragged.  Commonly 
the  individuals  comprising  this  chain  of  cocci  are  not  round, 
but  appear  flattened  on  the  sides.  The  chains  are  some- 
times short,  consisting  of  but  four  to  six  cells;  or,  again,  they 
may  be  much  longer,  and  extend  from  a  half  to  two-thirds 
the  way  across  the  field  of  the  microscope. 

Under  artificial  conditions  this  organism  sometimes  grows 
well,  and  can  be  cultivated  through  many  generations,  while 


STREPTOCOCCUS  PYOGENES  -    357 

at  other  times  it  rapidly  loses  its  vitality.  When  isolated 
from  the  diseased  area  upon  artificial  media  it  seems  to 
retain  its  vitality  for  a  longer  period  if  replanted  upon  fresh 
media  every  day  or  two  for  a  time;  but  if  the  first  generation 
be  transplanted  and  allowed  to  remain  upon  the  original 
medium  for  from  a  week  to  ten  days,  it  is  not  uncommon  to 
find  the  organism  incapable  of  further  cultivation. 

Under  no  conditions  is  its  growth  very  luxuriant. 

On  gelatin  plates  its  colonies  appear  after  forty-eight  to 
seventy-two  hours  as  very  small,  flat,  round  points  of  a 
bluish-white  or  opalescent  appearance.  They  do  not  cause 
liquefaction  of  the  gelatin,  and  in  size  they  rarely  exceed 
0.6-0.8  mm.  in  diameter.  Under  low  magnifying  power 
they  have  a  brownish  or  yellowish  tinge  by  transmitted 
light  and  are  very  finely  granular.  As  the  colonies  become 
older  their  regular  borders  may  become  slightly  irregular  or 
notched. 

In  stab-cultures  in  gelatin  they  grow  along  the  entire 
needle-track  as  a  finely  granular  line,  the  granules  represent- 
ing minute  colonies  of  the  organism.  On  the  surface  the 
growth  does  not  usually  extend  beyond  the  point  of 
puncture. 

On  agar-agar  plates  the  colonies  appear  as  minute  pearly 
points,  which  when  slightly  magnified  are  seen  to  be  finely 
granular,  of  a  light-brownish  .tinge,  and  regular  at  their 
margins. 

When  smeared  upon  the  surface  of  agar-agar  or  gelatin 
slants  the  growth  that  results  is  a  thin,  pearly,  finely  granular 
layer,  consisting  of  minute  colonies  growing  closely  side  by 
side.  Its  most  luxuriant  growth  is  usually  on  glyceriri-agar- 
agar  at  the  temperature  of  the  body  (37.5°  C.),  and  its 
least  on  gelatin  at  from  18°  to  20°  C. 


358      APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

On  nutrient  agar-agar  to  which,  under  aseptic  precautions, 
unheated,  defibrinated  or  whole  blood  has  been  added,  it 
often  causes  hemolysis,  i.  e.,  decolorization  of  the  blood  in 
the  neighborhood  of  the  growth.  Some  strains  of  the 
organism  when  thus  grown  are  surrounded  by  a  greenish 
zone,  Streptococcus  viridans;  others  by  a  perfectly  clear, 
colorless  zone,  Streptococcus  hemolyticus. 

On  blood  serum  its  colonies  present  little  that  is  character- 
istic; they  appear  as  small,  moist,  whitish  points,  from  0.6 
to  0.8  mm.  in  diameter,  that  are  slightly  elevated  above  the 
surface  of  the  serum.  They  do  not  coalesce  to  form  a  layer 
over  the  surface,  but  remain  as  isolated  colonies. 

On  potato  no  visible  development  appears,  but  after  a 
short  time  (thirty-six  to  seventy-two  hours)  there  is  a  slight 
increase  of  moisture  about  the  point  of  inoculation,  and 
microscopic  examination  shows  that  multiplication  of  the 
organisms  placed  at  this  point  has  occurred. 

In  milk  its  conduct  is  not  always  the  same,  some  cultures 
causing  a  separation  of  the  milk  into  a  firm  clot  and  colorless 
whey,  while  others  do  not  produce  this  coagulation.  The 
latter,  when  cultivated  in  milk  of  a  neutral  or  slightly 
alkaline  reaction,  to  which  a  few  drops  of  litmus  tincture 
have  been  added,  produce,  as  a  rule,  only  a  very  faint  pink 
color  after  twenty-four  hours  at  37°  C. 

In  bouillon  it  grows  as  tangled  masses  or  clumps,  which 
upon  microscopic  examination  are  seen  to  consist  of  long 
chains  of  cocci  twisted  or  matted  together. 

It  grows  best  at  the  temperature  of  the  body  (37.5°  C.), 
though  development  does  occur  at  the  ordinary  room-tem- 
perature. 

It  is  not  soluble  in  bile. 

It  ferments  some  of  the  carbohydrates,  notably  dextrose, 
maltose,  lactose  and  salicin,  but  not  mannite  or  inulin. 


STREPTOCOCCUS  PYOGENES  359 

It  is  a  facultative  anaerobe. 

It  stains  with  the  ordinary  aniline  dyes,  and  is  not  decolor- 
ized when  subjected  to  Gram's  method. 

It  is  not  motile.  Under  artificial  conditions  we  have  no 
reason  to  believe  that  it  enters  a  stage  in  which  its  resis- 
tance to  detrimental  agencies  is  increased.  In  the  tissues 
of  the  body,  however,  it  appears  to  possess  marked  vitality, 
for  it  is  not  rare  to  observe  recurrences  of  inflammatory 
conditions  due  to  this  organism,  often  at  a  relatively  long 
time  after  the  primary  site  of  infection  has  healed. 

Such  in  general  will  serve  to  identify  the  streptococci 
concerned  in  the  disease.. 

Streptococcus  pyogenes  is  the  organism  most  commonly 
found  in  rapidly  spreading  suppurations,  while  micrococcus 
aureus  is  most  frequently  found  in  circumscribed  abscess 
formations;  they  may  also  be  found  together,  and  these 
relationships  may  be  reversed  at  times. 

The  results  of  its  inoculation  into  the  tissues  of  lower 
animals  are  described  by  Rosenbach  and  Passet  as  pro- 
tracted, progressive,  erysipelatoid  inflammations;  and 
Fehleisen,  who  first  described  a  streptococcus  in  erysipelas 
that  is  closely  related  to  the  Streptococcus  pyogenes  under 
consideration,  stated  that  it  produced  in  the  tissues  of 
rabbits  (the  base  of  the  ear)  a  sharply  defined,  migratory 
reddening  without  pus  formation.  The  writer  encountered 
a  strain  of  this  organism  that  possessed  the  property  of 
inducing  erysipelas  when  introduced  into  the  skin  of  the 
ear,  and  disseminated  abscess  formation  when  injected  into 
the  circulation  of  rabbits.  In  one  animal  these  conditions 
appeared  simultaneously.  This  observation  has  an  impor- 
tant bearing  upon  the  question  concerning  the  identity  of 
streptococci  found  in  various  inflammatory  conditions,  such 


360     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

for  instance,  as  the  spreading  erysipelatoid  manifestations 
on  the  one  hand,  and  the  circumscribed  abscess  formations 
on  the  other. 

The  results  that  follow  upon  the  inoculation  of  animals 
with  cultures  of  streptococci  obtained  from  various  inflam- 
matory lesions  are,  as  a  rule,  inconstant.  At  times  cultures 
will  be  encountered  that  are  apparently  without  virulence, 
no  matter  how  tested;  while  again  cultures  from  other 
sources  exhibit  the  most  marked  pathogenic  properties, 
even  when  employed  in  almost  infinitesimal  quantities. 
Between  these  extremes  every  gradation  may  be  expected. 
The  virulence  of  a  culture  as  exhibited  upon  animals  under 
experiment  is  not  necessarily  proportional  to  the  intensity 
of  the  pathological  process  from  which  it  was  derived. 

In  general  it  may  be  said  that  the  virulence  of  strepto- 
coccus is  directly  proportionate  to  its  power  to  hemolyze 
blood.  That  is  to  say:  a  culture  that  actively  and  con- 
spicuously brings  about  the  laking  of  blood  with  complete 
decolorization  of  the  hemoglobin  is  more  apt  to  be  virulent 
than  one  devoid  of  that  property.  With  fluctuations  in 
such  hemolytic  function  there  are  corresponding  fluctuations 
in  virulence. 

There  is  never  any  certainty  of  faithfully  reproducing,  by 
inoculation  into  susceptible  animals,  the  pathological  lesion 
from  which  a  culture  of  the  organism  may  have  been  ob- 
tained. The  introduction  into  a  susceptible  animal  of  a 
culture  derived  from  either  a  spreading  phlegmon  or  an 
erysipelatous  inflammation  may  result  in  erysipelas,  general 
septicemia,  local  abscess-formation,  or,  as  said,  may  have 
no  effect  at  all.  Cultures  may  be  encountered  that  are 
pathogenic  for  one  susceptible  species  of  animals  and  not 
for  another. 


STREPTOCOCCUS  PYOGENES  361 

Under  the  ordinary  conditions  of  artificial  cultivation 
fully  virulent  varieties  of  Streptococcus  pyogenes  usually 
lose  their  virulence  after  a  short  time.  This  property  may 
sometimes  be  preserved  by  cultivation  upon  nutrient  gela- 
tin for  two  days  at  22°  C.,  keeping  the  cultures  after  this 
time  in  the  refrigerator,  and  transplanting  upon  fresh  gela- 
tin every  five  or  six  days;  or  by  growing  the  organism  in  a 
mixture  of  2  parts  of  horse  or  human  blood  serum  and  1 
part  of  nutrient  bouillon,  or  of  1  part  of  ascites  fluid  and 
2  parts  of  bouillon.  <  „  ^ 

Its  virulence  may  sometimes  be  increased  by  passage 
through  a  series  of  susceptible  animals. 

Variations  in  Streptococci. — The  obiquity  of  strepto- 
cocci and  their  frequent  relationship  to  pathological  condi- 
tions of  the  gravest  nature  combine  to  make  them  of  more 
than  passing  interest.  Our  knowledge  of  the  group  is  as 
yet  far  from  satisfactory,  yet  it  has  been  enhanced  in  many 
important  particulars  during  the  past  three  or  four  years. 

When  streptococci  are  encountered  in  the  various  diseased 
conditions  of  the  body  we  cannot  longer  content  ourselves 
with  the  fact  that  they  conform  to  the  commonly  accepted 
morphological  specifications  and  possess  the  customary 
gross  cultural  peculiarities  as  outlined  above.  We  have 
known  for  a  long  time  that  streptococci  vary  considerably 
in  a  number  of  particulars,  and  if  we  arbitrarily  decide  to 
call  a  given  streptococcus  a  typical  example  of  the  species 
we  shall  find  in  cultures  from  different  sources  very  many 
deviations  from  such  a  standard. 

According  to  the  nature  of  these  deviations  or  variations 
numerous  efforts  have  been  made  to  arrange  the  streptococci 
in  more  or  less  fixed  groups.  Such  efforts  have  not  been 
entirely  satisfactory  in  their  results,  though  they  have  con- 


362     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

tributed  helpful  information  to  our  general  comprehension 
of  the  subject. 

The  conception  that  streptococci  forming  long  chains 
are  more  often  pathogenic  than  those  forming  short  chains  is 
not  always  a  trustworthy  criterion ;  and  the  differences  in  the 
action  of  different  streptococci  upon  particular  ingredients 
of  special  culture  media  has  helped,  but  not  enough  for 
satisfactory  grouping.1 

The  discovery  that  the  majority  of  streptococci  associated 
with  serious  pathological  phenomena  have  the  power  of 
hemolyzing  blood,  while  others  do  not  possess  this  function 
is  a  great  step  in  advance,  but  unfortunately  for  the  sim- 
plicity of  the  matter  we  find  such  differences  among  those 
hemolyzing  or  hemolytic  streptococci  as  to  make  further 
subdivision  (classification)  of  the  group  desirable. 

With  this  in  mind  recent  studies  by  a  number  of  investi- 
gators have  contributed  information  of  the  greatest  practical, 
as  well  as  theoretical,  value. 

As  in  the  case  of  the  pneumococci  (which  see)  there  is 
now  an  agreement  of  opinion  that  differentiation  of  these 
closely  allied  varieties  of  pathogenic  organisms  is  best 
accomplished  through  specific  immunologic  reactions,  and 
to  this  end  the  agglutination  test  made  with  the  serum  of 
immunized  animals  seems  to  prove  trustworthy;  though  as 
yet  there  have  not  been  established  such  definite  groups 
or  types  as  has  been  done  with  the  pneumococci. 

If,  for  instance,  any  given  group  of  hemolytic  strepto- 
cocci be  obtained  from  different  diseases  and  an  animal 
be  rendered  immune  from  one  of  them  by  appropriate 
means,  the  serum  from  that  animal  will  certainly  agglu- 

1  See  The  Use  of  Blood  Agar  for  the  Study  of  Streptococci,  Monograph 
No.  9,  Rockefeller  Inst.  Med.  Research,  January  21,  1919. 


STREPTOCOCCUS  PYOGENES  363 

tinate  the  strain  of  streptococci  used  for  immunization. 
It  may  agglutinate  a  certain  number  of  the  other  hemo- 
lytic  varieties  but  it  is  unlikely  that  it  will  agglutinate 
all.  We  would  assume  then  that  etiologically  all  those 
agglutinated  by  that  serum  were  of  one  type,  while  all 
others  of  our  group  were  of  another  type  or  probably  types, 
and  so  we  might  continue  throughout  the  whole  group 
originally  selected  and  arrange  them  into  types  or  classes 
the  members  of  each  of  which  would  react  specifically  with 
its  homologous  serum  and  not  with  other  sera. 

The  object  of  such  grouping  is  more  far-reaching  than, 
that  of  simply  identifying  variations;  it  has  a  most  practical 
bearing  on  all  efforts  to  produce  antisera  that  may  be  used 
in  preventing  infection  or  in  curing  it  when  once  under  way. 

We  have  already  enough  evidence  to  justify  the  general 
statement  that  for  any  immune  serum  to  possess  protective 
or  curative  properties  for  a  bacterial  infection  the  animal 
from  which  it  is  obtained  must  have  been  immunized  with 
either  bacteria  direct  from  the  disease  against  which  the 
serum  is  to  be  used  or  with  types  closely  allied  to  them  in 
the  fundamental  infective  and  immunologic  characteristics. 
(Does  this  hold  for  such  infections  as  tetanus  and  diphtheria?) 

This  being  the  case  the  desirability  of  establishing  groups 
or  types  of  streptococci,  the  members  of  which  are  closely 
allied  in  these  particulars,  becomes  evident:  for  it  is  not 
practicable  in  efforts  to  treat  infections  along  these  lines 
to  always  immunize  animals  from  which  the  curative  serum 
is  to  be  obtained  with  the  organism  specifically  concerned 
in  the  case  under  consideration. 

Efforts  have  been  made  to  overcome  this  difficulty  by  the 
production  of  "polyvalent"  antisera,  i.  e.,  serum  from  ani- 
mals immunized  not  by  the  use  of  a  single  strain  of  strepto- 


364     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

cocci,  but  by  many,  the  idea  being  that  one  or  the  other 
of  the  component  streptococci  used  in  the  process  of  immun- 
ization may  be  identical,  or  sufficiently  nearly  so,  to  the  one 
concerned  in  the  infection  to  be  treated,  as  to  play  its  part 
in  the  production  of  the  desired  specific  component  of  the 
antiserum  so  obtained.  This  shot-gun-like  proceedure  some- 
times succeeds,  but  even  so,  the  element  of  uncertainty  is 
too  evident  to  justify  the  adherence  to  it  as  a  permanent 
method.  All  the  indications  point  to  the  substitution  of  a 
more  scientific,  a  more  logical  procedure  in  the  near  future, 
a  procedure  closely  allied  to  that  by  which  the  pneumo- 
cocci  have  been  grouped  and  from  which  so  much  light 
has  been  shed  upon  the  complicated  problem  of  pneumonia. 

Though  the  last  word  has  not  yet  been  said,  the  indi- 
cations are  that  in  erysipelas,  septic  sore-throat,  pleurisy, 
rheumatism,  scarlet  fever  anginas,  measles  sore-throat, 
postinfluenzal  pneumonia,  wound  infections,  etc.,  we  have 
streptococci  fundamentally  different  from  the  ubiquitous 
Streptococcus  pyogenes  as  commonly  described. 

Not  any  of  the  many  investigations  of  this  phase  of  the 
subject  appear  to  illustrate  more  clearly  the  possibilities 
and  practical  value  of  studies  upon  the  streptococci  than 
do  those  of  Havens,  conducted  in  U.  S.  Gen.  Hospital  No. 
12,  at  Biltmore,  N.  C.1 

Havens  undertook  to  classify  the  hemolytic  streptococci 
only,  as  there  is  now  a  general  agreement  that  the  non- 
hemolytic  varieties  are  of  but  subordinate  importance 
insofar  as  they  concern  infections  in  man. 

As  material  for  his  studies  292  different  strains  of  strepto- 
cocci were  used;  that  is  to  say,  there  were  that  number  of 

1  Jour.  Infect.  Dis.,  1919,  No.  4,  vol.  xxv. 


STREPTOCOCCUS  PYOGENES  365 

cultures,  each  from  a  different  individual.  These  indi- 
viduals came  from-  nearly  all  parts  of  the  country  and 
therefore  fairly  represented  conditions  to  be  found  through- 
out the  population  in  general.  The  physical  conditions 
of  the  persons  from  whom  the  cultures  were  obtained  were 
sufficiently  diverse  to x  indicate  the  comprehensive  nature 
of  the  investigation.  They  were: 

Throat  cultures: 

Healthy  carriers 80 

Acute  bronchitis 60 

Measles 9 

Sore-throat .      .      .      ....  30 

Pneumonia: 

Sputum 20 

Autopsy 21 

Empyema -.     >     .,     .      .      .-    .  67 

Gunshot  wounds         4 

Renal  infections          .      :      .      .      .      .      .      ...     -.   .  .  1 

Total 292 

By  selecting  at  random  from  the  292  cultures  three  that 
were  characteristic  in  their  gross  peculiarities  and  immun- 
izing animals  from  them,  it  was  found:  (1)  That  in  their 
agglutinating  reactions  these  cultures  were  identical;  and  (2) 
that  by  the  same  test  139  other  strains,  or  47  per  cent,  of  the 
whole  number,  proved  to  be  like  them,  while  the  remainder, 
153  strains,  failed  in  their  agglutinating  reactions  with  that 
serum  and  were  therefore  regarded  as  different.  Animals 
were  then  immunized  from  one  member  of  this  negative 
group  and  of  the  153  strains  in  that  group  54,  or  19  per 
cent.,  of  the  whole  number  agglutinated  with  the  serum 
from  this  immunized  animal.  Again  a  culture  of  those  that 
failed  to  agglutinate  in  the  second  test  was  chosen  and  a 
third  animal  rendered  immune  from  it.  With  the  serum 
from  that  animal  79  members  of  the  group,  or  27  per  cent., 
of  all  the  cultures  reacted  positively. 


366     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 


In  appropriately  high  dilutions  the  members  of  one  group 
did  not  react  with  the  serum  homologous  for  either  of  the 
other  groups. 

A  fourth  group,  containing  only  22  cultures,  gave  such 
irregularities  that  no  final  attempt  was  made  at  subgrouping. 

However,  of  the  total  of  292  strains  examined,  93  per  cent, 
could  be  definitely  arranged  by  specific  reactions  into  these 
groups;  while  7  per  cent.,  constituting  a  fourth  group,  was 
not  conclusively  investigated.  The  four  strains  of  hemo- 
lytic  streptococci  thus  established  grouped  themselves  about 
the  various  normal  and  pathological  conditions  from  which 
the  individual  cultures  were  obtained  according  to  the 
following  table: 

GROUPING  OF  292  STRAINS  OF  HEMOLYTIC  STREPTOCOCCI 

(HAVENS). 


Group  1. 
Per  cent. 

Group  2. 
Per  cent, 

Group  3. 
Per  cent. 

Group  4. 
Per  cent. 

Total  in 
per  cent. 

Number 
of  sources. 

Total    strains,    all 

sources   . 

47 

19 

27 

7 

100 

Throat  cultures: 

Healthy  carriers 

46 

20 

22 

12 

100 

80 

Acute  bronchitis 

48 

20 

23 

9 

100 

60 

Measles  . 

55 

45 

0 

0 

100 

9 

Tonsillitis      and 

sore-throat     . 

30 

10 

57 

3 

100 

30 

Bronchopneumonia 

Sputum  . 

40 

20 

30 

10 

100 

20 

Necropsy 

95 

0 

5 

0 

100 

21 

Emphysema    . 

42 

22 

30 

6 

100 

67 

Gunshot  wounds 

0 

0 

75 

25 

100 

4 

Renal  infections 

100 

0 

0 

0 

1 

From  this,  Havens  suggests  that  healthy  carriers  may  be 
the  source  of  supply  for  all  hemolytic  streptococci  causing 
disease. 


STREPTOCOCCUS  PYOGENES  367 

The  study  of  bronchopneumonias — though  small  in  num- 
ber— he  thinks  indicates  a  special  virulence  of  streptococci 
of  Group  I  in  this  disease. 

In  his  sore-throat  and  tonsillitis  cases  the  members  of 
Group  III  predominate  and  it  is  of  special  interest  to  note 
that  these  cases  all  came  from  one  hospital  ward  and  prob- 
ably therefore  had  a  common  origin. 

These  studies  further  show  that  the  specificity  of  this 
grouping  is  not  limited  to  the  agglutinating  reactions  of 
the  members  of  the  groups  but  is  still  further  demonstrated 
by  the  fact  that  in  vitro  the  streptococci  of  one  group  are 
killed  by  its  homologous  immune  serum,  while  such  serum  is 
without  germicidal  action  on  the  members  of  the  other  groups. 

By  analogous  proceedures  Tunnicliffe1  has  shown  that 
according  to  their  specific  serologic  reactions  the  strepto- 
cocci accountable  for  the  angina  of  scarlet  fever  and  those 
present  in  typical  cases  of  erysipelas  represent  distinct 
varieties  of  the  hemolytic  group  of  streptococci. 

The  bearing  of  all  this  on  efforts  to  produce  serum  for 
the  treatment  and  prevention  of  streptococcus  infections  is 
evident. 

We  can  no  longer  expect  the  serum  from  an  animal  immun- 
ized from  any  strain  of  streptococcus  taken  at  random  from 
whatever  source  to  be  effective.  It  may  be,  but  if  so  it  is 
only  by  chance.  Serum  A  may  be  expected  to  be  effective 
when  used  against  infection  caused  by  streptococcus  of  A 
group,  but  not  those  of  Group  B  and  vice  versa. 

Antistreptococcus  Serum. — Certain  animals  -  -  notably 
horses  and  asses — as  well  as  some  smaller  animals,  may  be 
rendered  immune  from  Streptococcus  pyogenes.  In  vary- 

1  Jour.  Am.  Med.  Assn.,  1920,  No.  20,  vol.  Ixxv,  p.  1339. 


368     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

ing  degrees  the  blood  serum  of  such  immunized  animals 
has  both  a  curative  and  a  prophylactic  influence  upon  the 
course  of  streptococcus  infection  in  human  beings. 

The  method  of  producing  the  antiserum  is,  in  general, 
to  inject  gradually  increasing  doses  of  virulent  Streptococcus 
pyogenes  (beginning  with  dead  cultures)-  into  the  tissues 
of  the  animal  until  its  blood  serum  is  found  to  have  an 
inhibiting  effect  upon  experimentally  produced  streptococcus 
infection  in  test  animals. 

Reports  upon  the  therapeutic  use  of  antistreptococcus 
serum  in  a  variety  of  streptococcus  infections  are  dis- 
cordant; some  authors  being  enthusiastic  as  to  its  curative 
value,  others  skeptical  or  actually  denying  to  it  such  virtues. 
The  reasons  for  these  divergent  opinions  are  now  pretty 
manifest  from  what  has  been  said  under  the  preceding 
heading  "Variations  in  Streptococci." 

THE   LESS   COMMON   PYOGENIC   ORGANISMS. 

The  organisms  that  have  just  been  described  are  com- 
monly known  as  the  "pyogenic  cocci"  of  Ogston,  Rosenbach, 
and  Passet,  and  up  to  as  late  as  1885  were  believed  to  be  the 
specific  factors  concerned  in  the  production  of  suppurative 
inflammations.  Since  that  time,  however,  there  has  been 
considerable  modification  of  this  view,  and  while  they  are 
still  known  to  be  the  most  common  causes  of  suppuration, 
they  are  also  known  to  be  not  the  only  causes  of  this  phe- 
nomenon. 

With  the  more  general  application  of  bacteriological 
methods  to  the  study  of  the  manifold  conditions  coming 
under  the  eye  of  the  physician,  the  surgeon,  and  the  patholo- 
gist, observations  are  constantly  being  made  that  do  not 


LESS  COMMON  PYOGENIC  ORGANISMS  369 

accord  with  the  earlier  ideas  upon  the  dependence  of  all 
forms  of  suppuration  on  invasion  by  the  pyogenic  cocci. 
There  is  an  abundance  of  evidence  to  justify  the  opinion 
that  a  number  of  organisms  not  commonly  classed  as  pyo- 
genic may,  under  certain  circumstances,  assume  this  property 
or  may,  in  fact,  have  pus  formation  as  one  of  the  common 
accompaniments  of  their  pathogenic  activities.  For  example : 

The  bacillus  of  typhoid  fever  has  been  found  in  pure 
culture  in  osteomyelitis  of  the  ribs,  in  acute  purulent  otitis 
media,  in  abscess  of  the  soft  parts,  in  the  pus  of  empyema, 
and  in  localized  fibrino-peritonitis,  either  during  its  course  or 
as  a  sequel  of  typhoid  fever. 

Bacillus  coli  communis  has  been  found  in  pure  culture  in 
acute  peritonitis,  in  liver-abscess,  in  purulent  inflammation 
of  the  gall-bladder  and  ducts,  and  in  appendicitis.  Welch1 
found  it  in  pure  culture  in  fifteen  different  inflammatory 
conditions. 

Micrococcus  lanceolatus  (pneumococcus)  has  been  found 
alone  in  abscess  of  the  soft  parts,  in  purulent  infiltration  of 
the  tissues  about  a  fracture,  in  purulent  cerebrospinal 
meningitis,  in  suppurative  synovitis,  in  acute  pericarditis, 
and  in  acute  inflammation  of  the  middle  ear. 

Organisms  simulating  bacterium  diphtheriticum  are  fre- 
quently encountered  in  large  numbers  in  the  pus  of  superfi- 
cial wounds,  and  especially  in  ulcerations  of  the  skin  and 
mucous  membranes. 

Moreover,  many  of  the  less  common  organisms  have  been 
detected  in  pure  cultures  in  inflammatory  conditions  with 
which  they  were  not  previously  thought  to  be  concerned, 
and  to  which  they  are  not  usually  related  etiologically. 

1  Conditions  Underlying  the  Infection  of  Wounds,  American  Journal  of 
the  Medical  Sciences,  November,  1891, 
24 


370      APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

In  consideration  of  such  evidence  as  this  it  is  plain  that 
we  can  no  longer  adhere  rigidly  to  the  opinions  formerly 
held  upon  the  etiology  of  suppuration,  but  must  subject 
them  to  modifications  in  conformity  with  this  newer  evi- 
dence. We  now  know  that  there  exist  bacteria  other  than 
the  "pyogenic  cocci,"  which,  though  not  normally  pyogenic, 
may  give  rise  to  tissue-changes  indistinguishable  from  those 
produced  by  the  ordinary  pus-organisms. 

Furthermore — of  organisms  not  classified  as  of  the 
"pyogenic  group,"  but  where  growth  in  the  tissues  is  always 
accompanied  by  pus  formation — one  may  mention  micro- 
coccus  gonorrhea,  micrococcus  intracellularis,  and  bacillus 
pestis  as  conspicuous  examples. 

MICROCOCCUS   GONORRHECE-ffi    (NEISSER),    1879. 

SYNONYM:    Gonococcus  Neisser,  Bumm,  1887. 

One  observes  upon  microscopic  examination  of  cover-slips 
prepared  from  the  pus  of  actue  gonorrhea  that  many  of  the 
pus-cells  contain  within  their  protoplasm  numerous  small, 
stained  bodies  that  are  usually  arranged  in  pairs.  Occasion- 
ally a  cell  is  seen  that  contains  only  one  or  two  pairs  of 
such  bodies;  again,  a  cell  will  be  encountered  that  is  packed 
with  them.  Occasionally  masses  of  these  small  bodies  will 
be  seen  lying  free  in  the  pus.  (See  Fig.  69.)  The  majority 
of  the  pus  cells  may  not  contain  them. 

These  small,  round,  or  oval  bodies  are  the  so-called 
"gonococci"  discovered  by  Neisser,  and  more  fully  studied 
subsequently  by  Bumm,  to  whom  we  are  indebted  for  much 
of  our  knowledge  concerning  them. 

As  the  name  implies,  this  organism  is  a  micrococcus,  and 
as  it  is  commonly  arranged  in  pairs  (flattened  at  the  sur- 


MICROCOCCUS  GONORRHCE&  371 

faces  in  juxtaposition)  it  is  often  designated  as  diplococcus 
of  gonorrhea.  It  is  always  to  be  found  in  gonorrheal  pus, 
and  often  persists  in  the  genital  discharges  and  secretions  far 
into  the  stage  of  convalescence.  It  is  not  present  -in  inflam- 
matory conditions  other  than  those  of  gonorrheal  origin. 

It  is  easily  detected  microscopically  in  the  secretions  of 
acute  gonorrhea.  In  secondary  lesions  and  in  very  old, 
chronic  cases  it  is  difficult  of  detection  and  frequently 

FIG.  69 

' 


f    *M  / 

ljj« 
i^e 

• 


Pus  of  gonorrhea,  showing  diplococci  in  the  bodies  of  the  pus-cells. 

eludes  all  efforts  to  find  it.  It  is  stained  by  the  ordinary 
methods,  but  perhaps  most  satisfactorily  with  the  alkaline 
solution  of  methylene-blue.  Most  important  as  a  differen- 
tial test  is  its  failure  to  stain  by  the  method  of  Gram.  (How 
does  this  compare  with  the  behavior  of  the  other  pyogenic 
cocci  when  treated  in  the  same  way?) 

It  does  not  grow  upon  ordinary  nutrient  media,  and  has 
only  been  isolated  in  culture  through  the  employment  of 
special  methods.  Its  growth  under  artificial  conditions 
seems  to  be  favored  by  some  particular  nutrient  substance 
that  is  supplied  by  blood  or  blood  serum,  and  in  many  of 


372     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

the  media  that  have  been  successfully  used  for  its  cultiva- 
tion this  substance  is  apparently  an  essential  constituent. 

It  was  first  isolated  in  culture  by  Bumm,  who  used  for 
this  purpose  coagulated  human  blood  serum  obtained  from 
the  placenta. 

Wertheim  improved  the  method  of  Bumm  by  using  a 
mixture  of  equal  parts  of  sterile  human  blood  serum  and 
ordinary  sterilized  nutrient  agar-agar,  the  latter  having 
been  liquefied  and  kept  at  50°  C.  until  after  the  mixture 
was  made,  when  it  was  allowed  to  cool  and  solidify. 

Other  investigators  have  substituted  for  human  blood 
serum  certain  pathological  fluids  from  the  human  body, 
such  as  ascites-fluid,  fluid  from  ovarian  cysts,  and  serous 
effusions  from  the  pleura  and  from  the  joint-cavities. 

The  method  used  by  Pfeiffer  for  the  cultivation  of  bac- 
terium influenzse  (see  that  method)  is  also  said  to  have  been 
successfully  employed. 

A  simple  medium  that  has  given  satisfactory  results  in 
our  hands  is  that  devised  by  Vedder.  It  consists  of  ordi- 
nary beef  infusion  agar  (1.5  per  cent,  agar)  to  which  1  per 
cent,  of  corn  starch  is  added.  The  medium  contains  neither 
sodium  chloride  nor  peptone  and  has  a  reaction  correspond- 
ing to  0.2  to  0.5  per  cent,  acid  to  phenolphthalein. 

Wassermann1  calls  attention  to  the  success  he  has  had 
in  cultivating  this  organism  upon  a  mixture  of  swine  serum 
and  nitrose,  the  latter  being  a  commercial  product  chemically 
known  as  casein-sodium  phosphate. 

The  preparation  of  the  medium  and  its  composition  are 
as  follows: 

In  an  Erlenmeyer  flask  mix  15  c.c.  of  swine  serum,  as 

1  Zeitschiift  fur  Hygiene  und  Infektionskrankheiten,  Bd.  xvii,  p.  298. 


MICROCOCCUS  GONORRHCEM  373 

free  as  possible  from  hemoglobin;  30  to  35  c.c.  of  water; 
2  to  3  c.c.  of  glycerin;  and  finally  0.8  to  0.9  gram  (i.  e., 
about  2  per  cent.)  of  nitrose.  This  is  boiled,  with  gentle 
agitation,  over  a  free  flame,  until  all  ingredients  are  dissolved 
and  the  cloudy  fluid  has  become  quite  clear.  After  such 
boiling  the  mixture  can  be  sterilized  by  steam  without 
precipitating  the  albumen,  and  may  then  be  kept  indefi- 
nitely ready  for  use. 

When  needed,  the  flask  and  its  contents  are  heated  to 
50°  C.;  from  six  to  eight  tubes  of  2  per  cent,  peptone- 
agar-agar  are  dissolved  by  boiling,  brought  to  50°  C., 
and  then  mixed  with  the  solution  in  the  flask  and  the  mass 
poured  into  Petri  dishes.  Upon  the  surface  of  this  serum- 
nitrose-agar  the  cultivation  is  to  be  conducted.  Wassermann 
lays  particular  stress  upon  two  points  that  are  essential  to 
success,  viz.?  the  preliminary  boiling  of  the  serum-nitrose 
mixture  before  steam  sterilization,  as  this  prevents  precipi- 
tation of  the  albumin;  and  the  necessity  of  having  both 
the  serum-nitrose  mixture  and  the  agar-agar,  to  be  mixed 
with  it,  at  not  over  50°  C.,  for  if  they  are  at  a  boiling  tem- 
perature when  mixed,  or  if  they  are  brought  to  the  boiling 
temperature  after  mixing,  the  albumin  will  be  precipitated 
notwithstanding  the  presence  of  the  nitrose,-  which  otherwise 
prevents  this. 

Wassermann  further  observes  that  some  samples  of  serum 
require  to  be  more  highly  diluted  with  water  than  in  the 
proportions  given  above;  that  the  agar-agar  should  be 
feebly,  but  distinctly,  alkaline  to  litmus,  causing  no  red- 
dening whatever  of  blue  litmus  paper;  and,  finally,  that 
the  Petri  dishes  containing  the  solidified  medium  on  which 
the  cultures  are  growing  are  best  kept  bottom  upward,  so 
as  to  prevent  water  of  condensation  collecting  on  the  surface. 


374     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

By  the  use  of  the  above  mediflm  he  has  cultivated  the  gono- 
coccus  from  about  one  hundred  different  cases. 

If  micrococcus  gonorrhceae  be  transplanted  from  the  origi- 
nal culture  to  either  glycerin-agar-agar  or  to  Loffler's  serum 
mixture,  a  growth  is  sometimes  observed,  more  often  in  the 
latter  than  in  the  former,  but  of  so  feeble  a  nature  that  these 
substances  cannot  be  regarded  as  suitable  for  its  cultivation 
and  certainly  not  for  its  direct  isolation  from  the  body.  As 
a  rule,  development  does  not  occur  on  glycerin-agar. 

Its  growth  is  favored  by  at  least  partial  anaerobic  con- 
ditions. 

Microscopic  examination  of  colonies  of  this  organism 
reveals  the  presence  of  a  diplococcus  somewhat  larger  than 
the  ordinary  pyogenic  cocci.  The  opposed  surfaces  of  the 
individual  cells  that  comprise  the  couplets  are  flattened  and 
separated  by  a  narrow  slit.  At  times  the  cocci  are  arranged 
as  tetrads. 

This  organism  cannot  be  grown  at  a  temperature  lower 
than  that  of  the  human  body,  and  cultures  that  have  been 
obtained  by  either  of  the  favorable  methods  are  said  to 
lose  their  vitality  when  kept  at  ordinary  room-temperature 
for  about  two  days. 

It  is  killed  in  a  few  hours  by  drying. 

Cultures  retain  their  vitality  under  favorable  conditions 
of  nutrition,  temperature,  and  moisture  for  from  three  to 
four  weeks. 

This  organism  is  without  pathogenic  properties  for 
monkeys,  dogs,  and  horses,  as  well  as  for  the  ordinary 
smaller  animals  used  for  this  purpose  in  the  laboratory. 

In  man  typical  gonorrhea  has  been  produced  by  the 
introduction  into  the  urethra  of  pure  cultures  of  this 
organism. 


MICROCOCCUS  GONORRHCEM  375 

In  addition  to  its  causal  relation  to  specific  urethritis, 
it  is  the  cause  of  gonorrheal  prostatitis  in  man,  of  gonorrheal 
proctitis  in  both  sexes,  and  of  gonorrheal  inflammation  of 
the  urethra,  of  Bartholin's  glands,  of  the  cervix  uteri,  and 
of  the  vagina  in  women  and  young  girls.  It  is  etiologically 
related  to  the  specific  conjunctivitis  (ophthalmia  neona- 
torum)  of  young  infants,  and  also  occasionally  to  ophthalmia 
in  adults. 

Secondarily,  it  is  concerned  in  specific  inflammations  of 
the  tubes  and  ovaries,  of  the  lymphatics  communicating 
with  the  genitalia,  of  the  serous  surfaces  of  joints,  and  of 
those  of  the  heart,  lungs,  and  abdominal  cavity. 

Other  species  of  micrococci  have  from  time  to  time  been 
described  as  occurring  in  the  pus  of  acute  urethritis  and  of 
other  purulent  inflammations.  Many  of  these  are  of  no 
significance.  Some  of  them  possess  peculiarities  that  might 
lead  to  confusion.  The  diplococcus  described  by  Heiman1 
has  certain  points  of  resemblance  to  the  gonococcus,  such 
as  its  location  in  the  ^bodies  of  pus-cells,  its  grouping  as 
diplococci,  its  size  and  general  appearance;  but  it  is  still 
readily  distinguished  from  the  gonococcus  by  its  retention 
of  color  when  treated  by  Gram's  method.  The  diplococcus 
detected  by  Bumm  in  puerperal  cystitis  is  likewise  often 
found  within  pus-cells,  but  it  is  readily  differentiated  from 
the  gonococcus  by  its  growth  upon  ordinary  nutrient  media. 
Micrococcus  intracellularis  of  Weichselbaum,  isolated 
from  the  sero-purulent  fluid  of  the  spinal  canal  in  cases  of 
epidemic  cerebrospinal  meningitis,  is  microscopically  also 
strikingly  like  the  gonococcus  as  it  is  seen  in  pus;  but, 
unlike  the  latter  organism,  may  be  cultivated  by  the  ordinary 

1  New  York  Medical  Record,  June  22,  1895. 


376     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

methods.  Micrococcus  catarrhalis,  so  often  seen  within  the 
bodies  of  pus  cells  in  the  nasal  discharges  of  acute  catarrh 
also  suggests  the  organism  under  consideration,  but  is -easily 
differentiated  by  its  growth  on  the  ordinary  culture  media. 

Summary  of  Distinguishing  Peculiarities. — Since  gonorrheal 
discharges  may  be  contaminated  with  pyogenic  cocci  other 
than  those  causing  the  specific  inflammation,  it  is  important 
in  efforts  to  identify  the  gonococcus  that  the  differential 
tests  be  borne  in  mind  and  put  into  practice.  The  gonococcus 
is  differentiated  from  the  commoner  pyogenic  organisms 
by  the  following  peculiarities. 

First,  it  is  practically  always  seen  in  the  form  of  diplococci, 
the  pair  of  individual  cells  having  the  appearance  of  two 
hemispheres,  with  the  diameters  opposed,  and  separated 
from  one  another  by  a  narrow,  colorless  slit.  (Is  this  the 
case  with  micrococcus  aureus  or  streptococcus  pyogenes?) 

Second,  in  gonorrheal  pus  it  is  nearly  always  within  the 
protoplasmic  bodies  of  pus-cells.  (How  does  this  compare 
with  the  conditions  found  in  ordinary  pus?) 

Third,  it  stains  readily  with  the  ordinary  staining  reagents, 
but  loses  its  color  when  treated  by  the  method  of  Gram.  (Treat 
a  cover-slip  from  ordinary  pus  by  this  method  and  note 
the  result.) 

Fourth,  it  does  not  develop  upon  any  of  the  ordinary 
media  used  in  the  laboratory;  while  the  common  pus- 
organisms,  with  perhaps  the  exception  of  the  streptococci, 
are  vigorous  growers  and  are  not  markedly  fastidious  as  to 
their  nutritive  medium. 

Fifth,  when  obtained  in  pure  culture  by  either  of  the 
special  procedures  noted  above,  its  cultivation  may  be 
continued  upon  the  same  medium;  but  growth  will  usually 
not  be  observed  if  it  is  transplanted  to  ordinary  nutrient 


MICROCOCCUS  INTRACELLULARIS  377 

gelatin,  agar-agar,  bouillon,  or  potato;  should  it  grow  under 
these  circumstances  its  development  will  be  very  feeble.  (Is 
this -the  case  with  common  pus-producers?) 

Sixth,  it  has  no  pathogenic  properties  for  animals,  while 
several  of  the  pyogenic  cocci,  notably  micrococcus  aureus 
and  streptococcus  pyogenes,  are  usually  capable  of  exciting 
pathological  conditions.  (This  is  less  commonly  true  of 
streptococcus  pyogenes  than  of  micrococcus  aureus.) 

Seventh,  it  has  the  power  of  fixing  complement,  and  this 
method  of  identification  is  of  particular  service  in  all  medico- 
legal  cases  as  well  as  in  other  obscure  cases  not  readily 
diagnosed  by  the  microscopic  and  cultural  methods.1 

MICROCOCCUS   INTRACELLULARIS    (WEICHSELBAUM), 
MIGULA,    1900. 

SYNONYMS:  Diplococcus  Intracellularis  Meningitidis,  Weichselbaum, 
1887;  Streptococcus  Intracellularis  (Weichselbaum),  Lehmann  and  Neu- 
mann, 1896. 

Of  the  several  organisms  mentioned  that  might  be  mis- 
taken for  the  gonococcus,  no  one  of  them  is  as  important 
as  that  concerned  in  the  causation  of  epidemic  cerebrospinal 
meningitis. 

This  organism,  described  by  Weichselbaum  in  1887  under 
the  name  "diplococcus  Intracellularis  meningitidis,"  was 
found  by  him  in  the  exudations  of  the  brain  and  spinal 
cord  in  six  cases  of  acute  cerebrospinal  meningitis. 

As  its  name  implies,  it  is  a  diplococcus,  practically  always 
seen  within  the  bodies  of  pus-cells  (polymorphonuclear 
leukocytes)  in  the  exudations  characteristic  of  this  disease. 
It  is  not  seen  within  the  other  cells  of  the  morbid  process. 

1  See  "Compliment-fixation;"  also  Schwartz  and  McNiel,  Am.  Jour. 
Mod.  Sc.,  cxliv,  p.  815. 


378     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

It  stains  readily  with  any  of  the  ordinary  aniline  dyes, 
but  is  decolorized  by  the  method  of  Gram.  It  is  conspicuous 
for  the  irregular  way  in  which  it  takes  up  the  dye,  some 
cells  in  a  preparation  (either  from  the  exudate  or  from  cul- 
tures) being  brightly  and  intensely  colored,  others  being 
much  less  so,  or,  indeed,  often  nearly  colorless.  There  is 
also  a  marked  variation  in  the  size  of  individual  cocci,  some 
being  normal,  others  being  apparently  swollen.  These 
latter  are  often  pale,  with  a  deeply  staining  center,  giving 
the  appearance  of  a  coccus  surrounded  by  a  capsule;  it 
is  not  improbable  that  these  are  degenerated.  The  ir- 
regularities here  noted  are  more  common  in  cultures 
than  in  fresh  exudates  from  acute  cases,  and  more  common 
in  old  than  in  young  cultures,  a  state  of  affairs  fully  explained 
by  the  self-digestion  (autolysis)  that  this  organism  is  known 
to  experience  under  conditions  of  artificial  cultivation. 

As  seen  in  cultures,  it  is  commonly  arranged  in  pairs  with 
the  individuals  flattened  at  the  surfaces  of  juxtaposition. 
Sometimes  it  is  seen  grouped  as  four  and  occasionally  as 
short  chains  of  three  or  four  cells,  but  never  as  long  chains. 
Its  size  is  that  of  the  common  pyogenic  micrococci,  and  its 
outline  and  arrangement  in  the  pus-cells  are  so  like  those  of 
the  gonococcus  that  the  figure  depicting  gonorrheal  pus 
answers  equally  well  to  illustrate  the  appearance  of  the 
exudate  from  acute  meningitis. 

Though  facultative,  still  its  parasitic  nature  is  so 
dominant  that  it  can  only  be  cultivated  with  difficulty 
and  uncertainty.  The  most  satisfactory  medium  for  its 
isolation  in  pure  culture  from  the  diseased  meninges  is 
coagulated  blood  serum  (LofEer's  mixture),  and  even  here 
one  is  not  successful  with  each  attempt.  So  uncertain  is 
its  growth  under  artificial  conditions  that  it  is  always  advis- 


MICROCOCCUS  INTRACELLULARIS  379 

able  to  inoculate  a  number  of  tubes  with  relatively  large 
quantities  of  the  exudate,  and  even  then  growth  often  occurs 
in  only  a  part  of  them,  notwithstanding  the  fact  that  on 
microscopic  examination  the  organism  may  have  been 
readily  detected  in  large  numbers  in  the  exudate.  Illus- 
trative of  this  difficulty,  the  following  experience  of  Council-  * 
man,  Mallory,  and  Wright  may  properly  be  quoted  i1 

"As  showing  the  difficulty  in  growing  the  organisms  in 
cultures  made  from  the  meninges  at  the  postmortem  exami- 
nation, ten  cultures  were  made  in  one  case  from  the  exuda- 
tion on  the  brain  and  six  from  the  cord,  cover-slip  exami- 
nations showing  abundant  organisms  in  the  cells.  Only 
two  of  the  cultures  from  the  brain  and  one  from  the  cord 
showed  a  growth.  As  a  rule,  the  organisms  were  more 
easily  obtained  in  cultures  made  from  the  acute  cases  than 
from  the  chronic." 

When  successfully  isolated  in  pure  culture  its  growth  is 
never  profuse  on  any  medium.  On  the  serum  mixture  of 
Loffler  the  isolated  colonies  appear  as  round,  viscid,  smooth, 
sharply  defined  points  that  may  attain  a  diameter  of  1  to 
1.5  mm.  There  is  no  liquefaction  of  the  medium.  Cultures 
from  very  acute  cases  occasionally  present  an  abundant 
growth  of  fine,  transparent  colonies  strongly  suggestive  of 
those  of  micrococcus  lanceolatus. 

On  glycerin-agar  the  colonies  are  round,  pearly,  trans- 
lucent, flat,  and  viscid  in  appearance.  They  tend  to  become 
confluent.  Under  low  magnifying  power  they  are  homo- 
geneous, semitransparent,  faintly  brownish,  with  well-defined 
smooth  margins.  On  plain  agar  the  growth  is  feeble  and 
uncertain. 

1  See  Epidemic  Cerebrospinal  Meningitis,  etc.,  Report  of  the  State  Board 
of  Health,  Mass.,  1898,  by  Councilman,  Mallory,  and  Wright. 


380      APPLICATION  OF  METHODS  OF 'BACTERIOLOGY 

Its  growth  in  bouillon  is  slow  and  uncertain.  It  does  not 
cause  clouding  of  the  fluid,  but  collects  at  the  bottom  of  the 
tube  as  a  scanty  grayish  sediment,  that  when  disturbed 
gives  the  impression  of  having  a  mucoid  consistency.  ;>  . 

It  does  not  grow  on  potato  and  causes  no  change  in  litmus- 
'  milk. 

It  grows  only  at  the  temperature  of  the  body,  and  can 
be  kept  growing  only  by  being  transplanted  to  fresh  media 
about  every  two  days,  and  even  then  growth  often  ceases 
after  a  comparatively  small  number  of  transplantations. 
If  from  a  fresh  growing  culture  a  number  of  tubes  be  inocu- 
lated and  kept  under  favorable  conditions  it  is  a  common 
experience  to  have  growth  on  only  a  part  of  them.  It  is 
sometimes  impossible  to  obtain  a  second  growth  on  agar-agar. 

In  addition  to  its  presence  in  the  meningeal  exudation 
of  epidemic  cerebrospinal  meningitis,  this  organism  may 
appear  as  a  secondary  invader  of  the  lung,  causing  more  or 
less  extensive  pneumonic  exudation;  of  the  joints;  the  ear; 
the  eye;  and  the  nose  and  throat.  Though  rarely,  its 
presence  in  the  circulating  blood  may  sometimes  be  demon- 
strated. 

Subcutaneous  inoculation  with  pure  cultures  has  usually 
no  effect.  Injections  into  the  great  serous  cavities  may  or 
may  not  result  in  serofibrinous  or  fibrinopurulent  inflam- 
mation. Positive  results  are  oftener  obtained  on  young 
guinea-pigs  weighing  about  150  grams,  than  on  larger, 
more  mature  animals.  Intravenous  inoculations  are  equally 
unsatisfactory,  though  the  results  depend  upon  the  original 
virulence,  the  age  of  the  culture  and  the  animal  selected. 
In  horses  toxic  symptoms  are  often  the  conspicuous  result  of 
this  mode  of  inoculation. 

The  only  successful  attempts  to  reproduce  the  morbid 


MICROCOCCUS  INTRACELLULARIS  381 

conditions  from  which  the  organism  is  obtained  are  those 
in  which  the  living  cultures  have  been  injected  directly 
into  the  meninges.  Weichselbaum  produced  congestion 
with  pus  formation  in  the  meninges  of  dogs  and  rabbits  by 
direct  injection  through  openings  made  in  the  skulls;  Coun- 
cilman, Mallory,  and  Wright  caused  the  death  of  a  goat  by 
the  injection  into  the  spinal  canal  of  1  c.c.  of  a  bouillon 
suspension  of  a  pure  culture  of  the  organism,  the  autopsy 
revealing  intense  congestion  of  the  meninges  of  both  brain 
and  cord,  with  slight  clouding  of  the  meninges  and  slight 
increase  of  meningeal  fluid,  and  Flexner1  succeeded,  through 
injections  of  cultures  into  the  spinal  canal  of  monkeys,  in 
causing  death  of  the  animals  with  inflammation  of  the  men- 
inges of  the  cord  and  brain. 

While  the  portal  of  entry  for  this  organism  to  the  system 
is  not  definitely  known,  it  is  still  of  importance  to  note  that 
it  often  makes  its  exit  from  the  body  by  way  of  the  organs 
that  are  secondarily  involved  and  that  open  to  without, 
as  the  ear,  nose,  eye  and  lungs. 

It  is  of  equal  importance  to  note  that  the  organism  is  of 
very  low  power  of  resistance,  being  destroyed  in  twenty- 
four  hours  by  direct  sunlight  and  by  drying  at  body-tem- 
perature, and  in  seventy-two  hours  by  drying  in  the  dark 
at  ordinary  room-temperature. 

For  the  diagnosis  of  epidemic  cerebrospinal  meningitis 
by  bacteriological  methods  it  is  essential  that  the  meningeal 
fluid  be  obtained  by  lumbar  puncture  during  the  most 
acute  stage  of  the  disease. 

Varieties. — As  in  the  case  of  the  pneumococci  and  strepto- 
cocci variations  are  observed  among  the  meningococci. 

1  Jour.  Exp.  Med.,  1907,  ix,  168. 


382     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

The  greatest  variations  are  seen  among  cultures  obtained 
from  healthy  persons  who  have  been  associated  with  cases 
of  meningitis,  i.  e.,  "the  carriers/'  while  the  least  degree 
of  variation  is  noted  in  cultures  direct  from  the  diseased 
tissues.  This  is  of  special  importance  in  indicating  the 
sources  from  which  cultures  should  be  derived  that  are  to 
be  used  in  the  immunization  of  animal  whose  serum  is  to  be 
employed  for  the  treatment  of  the  disease  in  man. 

Thus  far  two  main  groups  or  types  of  meningococci  have 
been  established,  and  there  is  a  possibility  of  further  sub- 
division of  these  types. 

Experience  in  this  field  shows  the  line  of  demarcation 
between  the  two  main  types  to  be  distinct,  but  for  the  pro- 
posed subtypes  it  is  less  sharp  than  that  for  the  other  organ- 
isms in  which  typing  has  succeeded,  that  is  to  say,  in  each 
of  the  subgroups  certain  individual  cultures  may  tend  to 
react  in  a  manner  suggesting  characters  common  to  members 
of  the  other  groups. 

In  establishing  the  groups  of  this  organism  the  method 
used  is  that  generally  employed,  i.  e.,  the  agglutinating 
reaction  with  homologous  immune  serum.  (See  paragraph 
on  "varieties"  in  articles  on  Pneumococci  and  Strepto- 
cocci.) 

ANTIMENINGITIS  SERUM.1  Flexner  has  demonstrated 
that  the  blood  serum  of  horses  and  of  goats  that  have 
received  repeated  subcutaneous  injections  of  cultures  of 
diplococcus  meningitidis  possesses  a  marked  restraining 
action  upon  the  course  of  meningitis.  This  is  true  not  only 
for  the  experimental  manifestations  of  the  disease,  but  for 
those  occurring  in  man  as  well.  The  analysis  of  about  400 

1  Flexner  and  Jobling,  Arch,  of  Pediatrics,  1908,  p.  747. 


PSEUDOMONAS  MRUGINOSA  383 

cases  of  true  epidemic  cerebrospinal  meningitis  in  man  in 
which  the  serum  was  used  shows  that  the  general  death 
rate  was  considerably  lower  than  that  following  any  other 
known  mode  of  treatment.  For  cases  treated  between  the 
first  and  third  days  of  the  disease  it  was  as  low  as  16.5  per 
cent.,  while  for  those  treated  as  late  as,  and  later  than  the 
seventh  day,  it  was  35  per  cent.  Between  these  figures 
the  rates  ran  from  20  to  25  per  cent.  For  success,  therefore, 
early  diagnosis  and  early  administrations  of  the  serum  are 
essential. 

There  is  no  agreement  of  opinion  as  to  how  antimeningitis 
serum  produces  its  favorable  results.  Several  suggestions 
have  been  offered:  It  may  stimulate  phagocytosis  and 
thus  lead  to  the  death  and  removal  of  the  meningococci;  it 
may  enter  into  destructive  union  with  the  specific  endo- 
toxin  of  the  meningococci;  or  it  may  act  directly  germicidal 
upon  the  organs  themselves. 

PSEUDOMONAS   -ffiRUGINOSA    (SCHROTER,    1872), 
MIGULA,   1900. 


SYNONYMS:      Bacterium   ^Eruginosum,   Schroter,    1872;    Bacillus 
ginosus,    Schroter,    1872;     Bacillus   Pyocyaneus,    Gessard,    1882;    Pseudo- 
monas  Pyocyanea,  Migula,  1896. 

Another  common  organism  that  may  properly  be  men- 
tioned at  this  place,  though  perhaps  not  strictly  pyogenic, 
is  a  pseudomonas  frequently  found  in  discharges  from 
wounds,  viz.,  pseudomonas  seruginosa,  or  bacillus  pyocyaneus 
or  "bacillus  of  green  pus,"  or  of  blue  pus,  or  of  blue-green 
pus,  as  it  is  by  custom  variously  designated.  Pseudomonas 
seruginosa  is  a  delicate  rod  with  rounded  or  pointed  ends. 
It  is  actively  motile;  does  not  form  spores.  As  seen  in 


384      APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

preparations  made  from  cultures,  it  is  commonly  clustered 
in  irregular  masses.  It  does  not  form  long  filaments,  there 
being  rarely  more  than  four  joined  end  to  end,  and  most 
frequently  occurs  as  single  cells. 

It  grows  readily  on  all  artificial  media,  and  gives  to  some 
of  them  a  bright-green  color  that  is  most  conspicuous  where 
it  is  in  contact  with  the  air.  This  green  color,  which  becomes 
more  and  more  marked  as  growth  advances,  is  not  seen  in 
the  growth  itself  to  any  extent,  but  is  diffused  through  the 
medium  on  which  the  organism  is  developing.  Ultimately 
this  color  becomes  much  darker,  and  in  very  old  cultures 
may  become  almost  black  (sometimes  very  dark  blue-green, 
at  others  brownish-black,  at  others  more  or  less  of  a  claret 
red). 

NOTE. — To  a  fresh  agar  culture  of  this  organism,  in 
which  the  green  coloration  of  the  medium  is  especially 
marked,  add  about  2  c.c.  of  chloroform.  Shake  gently,  and 
note  that  the  chloroform  extracts  a  blue  coloring-matter 
from  the  culture,  leaving  the  latter  more  or  less  yellow. 

Allow  the  chloroform  extract  to  stand  for  several  days; 
note  what  occurs;  how  do  you  account  for  it? 

Prepare  a  100  c.c.  Ehrlenmeyer  flask  with  75  c.c.  of 
sterile  bouillon  or  peptone  solution.  Inoculate  it  with  this 
organism  and  allow  it  to  stand,  without  shaking,  in  the 
incubator  at  body  temperature  for  about  a  week.  Note 
its  condition  on  removal.  Now  agitate  it  thoroughly  with 
air;  best  by  pouring  it  into  a  beaker  and  stirring  with  a 
glass  rod.  Note  what  now  occurs.  Now  abstract  with  5  c.c. 
of  chloroform — again  the  blue  extract  of  "pyoscyanin" 
is  obtained.  The  dirty  yellowish  or  reddish-yellow  color 
of  the  supernatant  fluid,  somewhat  fluorescent,  is  due  to 


PSEUDOMONAS  &RUGINOSA  385 

a  yellowish  pigment,  soluable  in  alcohol  and  water,  known 
as  "fluorescin." 

Cultivate  the  organism  in  one  or  another  of  the  synthe- 
sized media — Frankel's  modification  of  Uschinsky's  medium, 
for  instance : 

•    Water  distilled 1000  c.c. 

Asparagin 4  grams 

Ammonium  lactate 6  grams 

Hydrogen  Sod.  phosphate  (Na4H  Po2)      .      .      .  2  grams 

Sodium  chloride 5  grams 

Does  it  produce  any  color?  Is  chloroform  extract  of  such 
cultures  colored?  How  do  you  explain  the  result? 

Obtain  from  the  water  or  the  soil  an  organism  that  in 
several  particulars  suggests  B.  pyocyaneus,  namely,  bac- 
illus fluorescens  liquefaciens.  Repeat  the  foregoing  cul- 
tivations and  tests.  In  what  way  do  the  results  differ  from 
those  obtained  with  B.  pyoscyaneus? 

Make  two  bouillon  cultures  of  bacillus  fluorescens  lique- 
faciens. Place  one  in  the  incubator  and  keep  the  other  at 
room-temperature.  How  do  they  differ  at  end  of  forty-eight 
hours? 

Its  growth  in  gelatin-stab-cultures  is  accompanied  by 
liquefaction  and  the  diffusion  of  a  bright-green  color 
throughout  the  surrounding  unliquefied  medium.  As 
liquefaction  continues,  and  the  whole  of  the  gelatin  ulti- 
mately becomes  fluid,  the  green  color  is  confined  to  the 
superficial  layers  in  contact  with  the  air.  The  form  taken 
by  the  liquefying  portion  of  the  gelatin  in  the  earliest  stages 
of  development  is  somewhat  that  of  an  irregular  slender 
funnel.  (See  Fig.  70.) 

On  gelatin  plates  the  colonies  develop_rapidly;  they 
25 


386     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

are  not  sharply  circumscribed,  but  usually  present  at  first 
a  fringe  of  delicate  filaments  about  their  periphery.  (See 
Fig.  71.)  As  growth  progresses  and  liquefaction  becomes 
more  advanced  the  central  mass  of  the  colony  sinks  into 

FIG.  70 


FIG.  72 


FIG.  70. — Stab-culture  of  ps,  ceruginosa  in  gelatin  after  twenty-eight 
hours  at  22°  C. 

FIG.  71. — Colony  of  ps  ceruginosa  after  twenty-four  hours  on  gelatin  at 
20°-22°  C. 

FIG.  72. — Colony  of  ps.  ceruginosa  after  forty-two  hours  on  gelatin  at 
20°-22°  C. 


the  liquid,  while  at  the  same  time  there  is  an  extension  of 
the  colony  laterally.    At  this  stage  the  colony,  when  slightly 
magnified,    may    present    various    appearances,    the    most 
common  being  that  shown  in  Fig.  72. 
The  gelatin  between  the  growing  colonies  takes  on  a 


PSEUDOMONAS  &RUGINOSA  387 

bright  yellowish-green  color;  but  as  growth  is  comparatively 
rapid,  it  is  quickly  entirely  liquefied,  and  one  often  sees  the 
colonies  floating  about  in  the  pale-green  fluid. 

On  agar-agar  the  growth  is  dry,  sometimes  with  a  slight 
metallic  luster,  and  is  of  a  pale  gray  or  greenish-gray  color, 
while  the  surrounding  agar-agar  is  bright  green.  With 
time  this  bright  green  becomes  darker,  passing  into  blue- 
green,  and  finally  turns  almost  black. 

On  potato  the  growth  is  brownish,  dry,  and  slightly 
elevated  above  the  surface.  In  some  cultures  the  potato 
about  the  line  of  growth  becomes  green;  in  others  this 
change  is  not  so  noticeable.  With  many  cultures  a  peculiar 
phenomenon,  consisting  of  a  change  of  color  from  brown  to 
green,  may  be  produced  by  lightly  touching  the  growth  with 
a  sterile  platinum  needle.  The  change  occurs  only  at  the 
point  touched.  It  is  best  seen  in  cultures  that  have  been 
kept  in  the  incubator  for  from  seventy-two  to  ninety-six 
hours.  It  occurs  in  from  one  to  three  minutes  after  touching 
with  the  needle,  and  may  last  for  from  ten  minutes  to  a  half- 
hour.  This  is  the  "  chameleon  phenomenon"  of  Paul  Ernst. 

In  bouillon  the  green  color  appears,  and  the  growth  is 
seen  in  the  form  of  delicate  flocculi.  A  very  delicate  my  co- 
derma  is  also  produced.  As  growth  progresses,  the  bouillon 
becomes  darker  and  darker  in  color,  and  more  or  less  fluores- 
cent, until  it  finally  is  about  comparable  in  this  respect  to 
crude  petroleum;  at  the  same  time  it  assumes  a  peculiar 
ropiness,  and  very  old  cultures  (four  to  six  weeks  in  the  incu- 
bator) may  attain  about  the  consistency  of  raw  egg-albumen. 
This  is  due  to  the  production  of  a  substance  closely  allied, 
chemically  speaking,  to  mucin.  Whether  it  is  a  metabolic 
product  or  one  resulting  from  the  degeneration  or  the  auto- 
digestion,  so  to  speak,  of  the  bacteria,  cannot  now  be  said; 


388     APPLICATION  OF   METHODS   OF   BACTERIOLOGY 

at  all  events,  in  cultures  presenting  this  peculiarity  very 
few  bacteria  of  normal  appearance — indeed,  very  few 
bacteria  at  all — are  to  be  seen  on  microscopic  examination. 

In  milk  it  causes  an  acid  reaction,  with  coincident  coagula- 
tion of  the  casein. 

On  blood  serum  and  egg-albumen  its  growth  is  accom- 
panied by  liquefaction.  The  growth  on  coagulated  egg- 
albumen  is  seen  as  a  dirty-gray  deposit  surrounded  by  a 
narrow  brownish  zone;  the  remaining  portion  of  the  medium 
is  bright  green  in  color.  As  the  culture  becomes  older  the 
green  may  give  way  to  a  brown  discoloration. 

In  peptone  solution  it  causes  a  bluish-green  color.  In 
one  of  four  cultures  from  different  sources  we  observed  the 
production  of  a  distinct  blue  color.  In  another  specimen 
the  fluid  was  of  a  distinct  wine  red  color,  after  five  days  at 
body  temperature. 

It  produces  indol. 

It  stains  with  the  ordinary  dyes,  and  its  flagella  may 
readily  be  demonstrated  by  appropriate  methods  of  staining. 

It  is  an  active  producer  of  a  proteolytic  enzyme  that  may 
readily  be  separated  and  its  digestive  properties  observed 
by  the  following  simple  method:  Prepare  a  bouillon  culture 
of  about  70  to  80  c.c.  volume,  and  allow  it  to  grow  at  37° 
to  38°  C.  for  four  or  five  days.  Filter  through  a  Berkefeld 
filter  into  a  sterile  receiver.  Under  aseptic  precautions 
decant  the  filtrate  into  sterile  test-tubes,  about  7  c.c.  to 
each  tube.  Then  under  aseptic  precautions  make  the 
following  tests:  To  one  tube  add  a  small  bit  of  hard-boiled 
egg  (about  one-half  the  size  of  a  pea)  and  place  in  an  incu- 
bator. Render  another  tube  slightly  acid  with  dilute 
hydrochloric  acid,  and  add  a  bit  of  the  white  of  egg  to  it 
also.  Do  the  results  differ? 


PSEUDOMONAS  MRUGINOSA  389 

Heat  another  tube  to  80°  C.  for  fifteen  minutes,  and 
repeat  the  experiment.  Has  the  heating  had  any  effect  ? 

To  another  tube  add  carbolic  acid  to  the  extent  of  2  or  3 
per  cent.  Is  the  digestive  activity  of  the  solution  modified? 

To  two  ordinary  tubes  of  gelatin  add  carbolic  acid 'until 
it  is  present  to  the  extent  of  0.25  per  cent,  in  each  tube. 
Solidify  the  gelatin  in  one  tube  in  the  upright  position;  let 
that  in  the  other  remain  fluid.  On  the  surface  of  the  former 
pour  0.5  c.c.  of  the  pyocyaneus  filtrate,  and  mark  the  point 
of  contact  between  the  gelatin  and  filtrate.  To  the  other 
tube  add  a  similar  amount  of  filtrate,  mix  thoroughly,  and 
solidify  in  a  glass  of  cold  water. 

At  the  end  of  eighteen  to  twenty  hours  note  result.  Is 
it  possible  to  solidify  again  the  gelatin  through  which  the 
filtrate  was  mixed,  by  placing  the  tube  in  cold  water? 

Do  the  activities  of  this  enzyme  suggest  those  of  any  of 
the  enzymes  encountered  in  the  animal  body?  Which?  and 
Why? 

Extract  with  chloroform  a  six  days'  old  bouillon  culture 
of  this  organism.  In  which  portion  of  the  liquid  so  extracted 
is  the  proteolytic  ferment  contained,  the  chloroform  extract 
or  the  supernatant  fluid? 

Mix  slowly  a  two  weeks'  old  bouillon  culture  of  this 
organism,  grown  at  body  temperature,  with  six  times  its 
volume  of  absolute  alcohol.  Allow  to  stand  over  night. 
Filter.  Redissolve  the  precipitate  in  a  few  c.c.  (5  or  6),  of 
physiological  salt  solution.  In  the  meantime  evaporate  the 
alcohol  filtrate  to  dryness  at  a  temperature  not  exceeding  40° 
C.,  and  redissolve  the  sedement  in  5  or  6  c.c.  of  physio- 
logical salt  solution.  Test  both  of  these  solutions  on  car- 
bolized  gelatin  for  proteolytic  activity.  What  are  the 
results  and  how  are  they  explained? 


390     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

Inoculation  into  Animals. — As  a  rule,  cultures  of  this 
organism  obtained  directly  from  the  discharges  of  the  wound 
are  capable,  when  introduced  into  animals,  of  producing 
diseased  conditions;  but  cultures  kept  on  artificial  media 
for  a  long  time  may  in  part,  or  completely,  lose  this  power. 

When  guinea-pigs  or  rabbits  are  inoculated  subcutaneously 
with  1  c.c.  of  virulent  fluid  cultures  of  this  organism,  death 
usually  results  in  from  eighteen  to  thirty-six  hours.  At 
the  seat  of  inoculation  there  are  found  an  extensive  purulent 
infiltration  of  the  tissues  and  a  marked  zone  of  inflammatory 
edema. 

When  introduced  directly  into  the  peritoneal  cavity  the 
results  are  also  fatal,  and  at  autopsy  a  genuine  fibrinous 
peritonitis  is  found.  There  is  usually  an  accumulation  of 
serum  in  both  the  peritoneal  and  pleural  cavities.  At 
autopsies  after  both  methods  of  inoculation  the  organisms 
will  be  found  in  pure  cultures  in  the  blood  and  internal 
viscera. 

When  animals  are  inoculated  with  small  doses  (less  than 
1  c.c.  of  a  bouillon  culture)  of  this  organism  death  may  not 
ensue,  and  only  a  local  inflammatory  reaction  (abscess 
formation)  may  be  set  up.  In  these  cases  the  animals  are 
usually  protected  from  subsequent  inoculation  with  doses 
that  would  otherwise  prove  fatal. 

Most  interesting  in  connection  with  pseudomonas  ceru- 
ginosa  is  the  statement  of  Bouchard,  and  of  Charrin  and 
others,  that  its  products  possess  the  power  of  counteract- 
ing the  pathogenic  activities  of  bacterium  anthracis.  That 
is  to  say,  if  an  animal  be  inoculated  with  a  virulent  anthrax 
culture,  and  soon  after  be  inoculated  with  a  culture  of  pseudo- 
monas oeruginosa,  the  fatal  effects  of  the  former  inoculation 


BACILLUS  PEST  IS  391 

may  be  prevented.  Emmerich  and  Low1  are  inclined  to 
attribute  this  to  the  direct  bacteriolytic  action  of  the  enzymes 
upon  the  anthrax  bacteria  introduced  into  the  tissues. 

In  the  literature  upon  the  green-producing  organisms  that 
have  been  found  in  inflammatory  conditions  several  varieties 
—believed  to  be  distinct  species — have  been  described;  but 
when  cultivated  side  by  side  their  biological  differences  are 
seen  to  be  so  slight  as  to  render  it  probable  that  they  are 
but  modifications  of  one  and  the  same  species. 

BACILLUS    PESTIS,   YERSIN,    1894.      THE   BACILLUS    OF 
BUBONIC   PLAGUE. 

Before  passing  from  the  subject  of  suppuration  it  may 
not  be  inappropriate  to  call  attention  to  the  light  that 
modern  methods  of  investigation  have  shed  upon  the  etiology 
of  bubonic  plague,  an  epidemic  disease  characterized  by 
suppuratipn  of  the  lymphatic  glands,  and  accompanied  by 
a  very  high  rate  of  mortality,  especially  when  the  infection 
involves  the  lungs,  as  is  sometimes  the  case. 

This  pestilence,  probably  endemic  in  certain  sections  of 
the  Orient,  is  one  of  the  most  conspicuous  epidemic  diseases 
of  history.  Since  early  in  the  Christian  era  epidemics  and 
pandemics  of  plague  have  made  their  appearance  in  Europe 
at  different  times.  During  and  for  a  time  after  the  Middle 
Ages  it  was  more  or  less  frequent  in  India,  China,  Arabia, 
Northern  Africa,  Italy,  France,  Germany,  and  Great  Britian. 
In  history  it  is  variously  known  as  the  "Justinian  Plague" 
of  the  sixth  century,  the  "Black  Death"  of  the  fourteenth 
century,  and  the  "Great  Plague  of  London"  of  the  seven- 

1  Munchener  med.  Wochenschrift,  1898,  No.  40;  Centralblatt  fiir  Bakter- 
iologie  und  Parasitenkunde,  1899,  Abt.  i,  No.  1,  p.  33. 


392      APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

teenth  century,  though  it  is  difficult  to  say  to  what  extent 
these  outbreaks  were  uncomplicated  manifestations  of 
genuine  bubonic  plague.  During  the  existence  of  the  Jus- 
tinian Plague  10,000  people  are  said  to  have  died  in  Con- 
stantinople in  a  single  day,  and  Hecker  estimates  that  during 
the  pandemic  of  the  Black  Death  25,000,000  people  (a 
quarter  of  the  entire  population  of  Europe)  succumbed  to 
the  disease.  During  the  Great  Plague  of  London  (1664-65) 
the  total  mortality  for  one  year  was  68,596,  out  of  an  esti- 
mated population  of  460,000  souls. 

It  is  not  surprising  to  learn  that  it  was  to  guard  against 
the  plague  that  quarantine  regulations  were  first  estab- 
lished. 

The  first  and  certainly  the  most  exact  information  con- 
cerning the  exciting  cause  and  the  pathology  of  the  plague 
was  furnished  by  investigations  of  Yersin,  of  Kitasato,  and 
of  Aoyama,  conducted  during  the  epidemic  of  1894  in  Hong 
Kong,  China;  although  since  then  numerous  other  inves- 
tigators have  made  additional  important  contributions  to 
our  knowledge  of  the  subject.  The  results  of  these  studies 
demonstrate  that  bubonic  plague  is  an  infectious,  not 
markedly  contagious  disease  (except  in  the  case  of  the 
pulmonic  variety),  that  depends  for  its  existence  upon  the 
presence  in  the  tissues  of  a  specific  microorganism — the 
so-called  plague  or  pest  bacillus. 

This  organism  is  described  as  a  short,  oval  bacillus,  usually 
seen  single,  sometimes  joined  end  to  end  in  pairs  or  threes, 
less  commonly  as  longer  threads.  It  stains  more  readily 
at  its  ends  than  at  its  center.  It  is  sometimes  capsulated; 
is  non-spore-forming;  is  aerobic,  and  is  non-motile.  It  is 
found  in  large  numbers  in  suppurating  glands.  (Fig.  73.) 
It  is  also  to  be  detected  in  the  blood,  spleen,  lungs,  liver, 


BACILLUS  PESTIS 


393 


kidneys,  walls  of  the  stomach  and  intestines,  urine,  and 
intestinal  contents  of  fresh  cadavers;  and  during  life  in  the 
blood,  expectorations,  feces,  and  urine  of  persons  sick  of  the 
disease.  From  these  findings  the  infection  is  obviously  a 
septicemia. 

FIG.  73 
A 


'<*/"'  C     < 

^  '  "  '41 


Bacillus  of  bubonic  plague:     A,   in  pus  from  suppurating  bubo;   B,   the 
bacillus  very  much  enlarged  to  show  peculiar  polar  staining. 


It  is  negative  to  the  Gram  method  but  stains  readily  with 
the  ordinary  aniline  dyes.  It  may  be  cultivated  upon 
ordinary  nutrient  media,  although  preference  is  given  by 
some  to  a  neutral  or  slightly  alkaline  2  per  cent,  peptone 
solution  containing  from  1  to  2  per  cent,  of  gelatin. 

The  most  favorable  temperature  for  its  growth  is  between 


394     APPLICATION  OF  METHODS  OF   BACTERIOLOGY 

36°  and  39°  C.  Its  colonies  on  glycerin-agar-agar  and  on 
coagulated  blood-serum  are  described  as  iridescent,  trans- 
parent, and  whitish.  On  gelatin  at  18°-20°  C.  it  develops 
as  small,  sharply  defined,  white  colonies  without  liquefaction 
of  the  medium.  In  stab-cultures  it  develops  both  on  the 
surface  and  along  the  track  of  the  needle.  Its  growth  is 
slow.  It  does  not  cause  a  diffuse  clouding  of  bouillon,  but 
grows  rather  as  irregular,  flocculent  clumps  that  adhere  to 
the  sides  or  sink  to  the  bottom  of  the  vessel,  leaving  the  fluid 
clear.  It  shows  but  limited  growth  on  potato.  It  does  not 
ferment  glucose  with  production  of  gas,  nor  does  it  form 
indol.  It  coagulates  milk. 

This  organism  is  killed  by  drying  at  ordinary  room-tem- 
perature in  four  days.  It  is  killed  in  three  or  four  hours 
by  direct  sunlight.  It  is  destroyed  in  a  half  hour  by  80° 
C.,  and  in  a  few  minutes  by  100°  C.  (steam).  It  is  killed  in 
one  hour  by  1  per  cent,  carbolic  acid  and  in  two  hours  by 
1  per  cent,  milk  of  lime.1 

It  is  pathogenic  for  rats,  mice,  guinea-pigs,  ground  squir- 
rels, rabbits,  hogs,  horses,  monkeys,  cats,  chickens,  and 
sparrows.  Pigeons,  hedgehogs,  and  frogs  are  immune,  and 
dogs  and  bovines  are  apparently  so.2 

Animals  succumb  to  subcutaneous  inoculation  in  from  two 
to  three  days.  According  to  Yersin,  the  site  of  subcutaneous 
inoculation  becomes  edematous  and  the  neighboring  lym- 
phatics are  enlarged  in  a  few  hours.  After  twenty-four  hours 
the  animal  is  quiet,  the  hair  is  rumpled,  tears  stream  from  the 
eyes,  and  later  convulsions  set  in,  which  last  till  death.  The 

1  See  Viability  of  the  Bacillus  Pestis,  by  M.  J.  Rosenau,  U.  S.  Marine- 
Hospital  Service,  Bulletin  No.  4,  of  the  Hygienic  Laboratory,  U.  S.  M.-H., 
Washington,  D.  C.,  1901. 

JNuttall,  Centralblatt  fiir  Bakteriologie  und  Parasitenkunde,  1897, 
Abt.  1,  Bd.  xxii,  S.  97. 


BACILLUS  PESTIS  395 

results  found  at  autopsy  are:  blood-stained  edema  at  the 
site  of  inoculation,  reddening  and  swelling  of  the  lymphatic 
glands,  bloody  extravasation  into  the  abdominal  walls,  serous 
effusion  into  the  pleural  and  peritoneal  cavities;  the  intes- 
tine is  occasionally  hyperemic,  the  adrenal  bodies  congested, 
and  the  spleen  enlarged,  often  being  studded  with  grayish 
points,  suggestive  of  miliary  tubercles.  The  plague,  or  pest, 
bacillus  is  detected  in  large  numbers  in  the  local  edema,  the 
lymph  glands,  the  blood,  and  the  internal  organs. 

As  is  the  case  in  general  with  the  group  of  hemorrhagic 
septicemia  bacteria,  the  members  of  which  it  resembles  in 
certain  other  respects,  when  death  does  not  result  promptly 
after  infection  there  is  usually  only  local  evidence  of  the 
inoculation,  the  distribution  of  the  microorganisms  through- 
out the  body  being  considerably  diminished. 

Animals  that  survive  inoculation  with  this  organism 
usually  exhibit  a  certain  degree  of  immunity  from  subsequent 
infection. 

Nuttall1  notes  that  feeding  experiments  have  resulted 
in  fatal  infection  in  gray  and  white  rats,  house-  and  field- 
mice,  guinea-pigs,  rabbits,  hogs,  apes,  cats,  chickens,  sparrows, 
and  flies.  He  also  calls  attention  to  the  fact  that  flies  may 
live  for  several  days  after  being  infected  with  this  organism, 
and  if  at  liberty  to  fly  about  may  infect  persons  or  foodstuffs 
on  which  they  alight  or  fall. 

All  opinions  and  investigations  agree  in  that  the  flea  is  the 
most  common  and  important  of  the  agents  of  transmission, 
carrying  the  disease  from  man  to  animals  (rodents,  rats  in 
particular)  and  from  animals  to  man. 

The  bacilli  apparently  lose  their  virulence  after  long-con- 

1  Loc  cit. 


396     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

tinued  cultivation  under  artificial  conditions,  and  it  is  said 
that  from  slowly  developing,  chronic  buboes  non-virulent 
or  feebly  virulent  cultures  are  often  obtained.  Variations 
in  the  degree  of  virulence  have  been  observed  in  different 
colonies  from  the  same  source.  Virulence  is  said  to  be 
accentuated  by  passing  the  organism  through  a  series  of 
susceptible  animals. 

It  has  been  observed  that  in  the  suppurating  lymphatic 
glands  of  man  a  variety  of  organisms  may  be  present,  but 
among  them  are  always  the  plague  bacilli.  Occasionally 
micrococci  predominate.  In  these  cases  of  mixed  infection 
the  pest  bacilli  are  said  to  stain  less  intensely  with  alkaline 
methylene-blue  than  do  the  streptococci,  and  more  intensely 
than  do  the  micrococci  that  are  present.  Also,  in  this  event, 
the  streptococci  retain  the  Gram  stain,  while  the  pest  bacilli 
do  not  and  the  staphylococci  may  or  may  not. 

It  is  the  opinion  of  Aoyama  that  the  suppuration  of  the 
glands  is  not  caused  by  the  plague  bacillus,  but  is  rather  the 
result  of  the  action  of  the  pyogenic  cocci  with  which  it  is 
so  often  associated.  He  does  not  regard  either  the  air- 
passages  or  the  alimentary  tract  as  frequent  portals  of  infec- 
tion. Wilm,  on  the  contrary,  is  inclined  to  regard  the 
alimentary  tract  as  a  frequent  portal  of  infection;1  and  there 
are  numerous  opinions  that  in  the  pulmonic  type  of  plague, 
its  most  fatal  manifestation,  infection  is  always  by  way 
of  the  respiratory  tract. 

The  order  in  which  the  lymphatics  manifest  disease  ap- 
pears to  depend  upon  the  location  of  the  primary  infection. 
That  is  to  say,  if  it  is  upon  the  lower  extremities,  the  super- 
ficial and  deep  inguinal  glands  are  the  first  to  show  signs 

1  Wilm,  Hyg.  Rundschau,  1897,  p.  217. 


BACILLUS  PESTIS  397 

of  the  disease;  while  if  infection  occurs  via  the  hands  and 
arms,  the  buboes  appear  first  in  the  axillary  region.  As  a 
rule,  the  wound  through  which  infection  is  received  shows 
little  or  no  inflammatory  reaction. 

The  blood  of  patients  convalescing  from  plague  has  an 
agglutinating  action  upon  fluid  cultures  of  the  plague  bacillus 
analogous  to  that  observed  when  the  blood-serum  of  typhoid 
or  of  cholera  patients  is  mixed  with  similar  cultures  of  the 
typhoid  or  the  cholera  bacillus.  (See  Agglutinins). 

Protective  Inoculation;  Vaccination. — Active  immunization 
from  plague  infection  by  protective  inoculation  has  been 
variously  attempted;  by  subcutaneous  or  intramuscular 
injection  of  old  bouillon  cultures  of  bacillus  pestis  that  had 
been  killed  by  heat;  by  similar  injections  of  emulsions  made 
from  agar-agar  cultures  of  different  ages  suspended  in 
isotonic  salt  solution  and  likewise  killed  by  heat;  by  the 
injection  of  determined  amounts  of  extractives  from  plague 
bacilli;  by  the  injection  of  mixtures  of  dead  plague  bacilli 
and  plague  immune  serum;  by  injection  of  the  filtrate  from 
fluid  cultures  of  the  organism;  by  the  injections  of  peri- 
toneal exudates  and  organ  extracts  of  animals  infected  with 
plague;  and  by  the  injection  of  attenuated  living  cultures 
of  the  organism.  For  the  most  part  these  efforts  have  been 
experimental,  that  is  to  say,  they  have  been  made  upon 
animals  susceptible  to  plague  infection,  notably  guinea-pigs 
and  monkeys.  In  the  problem  of  protecting  human  beings 
from  plague,  dead  cultures  have  been  used  practically  to 
the  exclusion  of  all  other  methods.  The  method  of  Haffkine1 
has  enjoyed  more  favor  than  any  of  the  others,  though  it 
is  difficult  to  determine  its  protective  value  with  any  degree 
of  exactness.2  This  method  consists  in  the  subcutaneous 

1  British  Med.  Jour.,  1897,  No.  12. 

2  Bull,  de  1'Institut  Pasteur,  1906,  No.  4,  p.  825. 


398     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

injection  of  from  0.5  c.c.  to  7.0  c.c.  of  a  six  weeks'  old, 
specially  prepared  bouillon  culture  of  bacillus  pestis  that 
had  been  killed  by  exposure  to  65°  C.  for  one  hour.  Some- 
times the  smaller,  sometimes  the  larger  doses  are  indicated; 
sometimes  a  single  injection  is  given,  sometimes  several  are 
repeated  at  shorter  or  longer  intervals  according  to  circum- 
stances. The  injections  are  followed  by  both  local  and  con- 
stitutional reactions,  varying  in  the  degree  of  intensity  and 
length  of  duration  with  different  individuals.  The  immunity 
resulting  is  said  to  be  established  fairly  promptly  and  to  last 
for  six  weeks  and  longer.  The  investigations  of  the  Indian 
Plague  Commission  justify  the  conclusion  that  both  mor- 
bidity and  mortality  for  Plague  is  less  among  the  inoculated 
than  among  the  uninoculated. 

In  so  far  as  experiments  upon  animals  and  observations 
upon  human  beings  afford  positive  light  on  this  subject, 
the  protective  inoculations  protect  only  against  the  bubonic 
type  of  plague  and  are  practically  without  influence  in 
preventing  the  pulmonary  or  pneumonic  manifestation. 

The  comprehensive  critical  review  of  this  subject  made 
by  Strong1  led  him  to  the  same  conclusion  as  that  of  Kolle 
and  Otto:2  that  the  most  effective  protection  from  plague 
is  that  afforded  by  the  injection  of  attenuated,  living  cul- 
tures. Tests  made  upon  monkeys  and  guinea-pigs  demon- 
strated this  method  to  be,  in  round  numbers,  three  times 
as  effective  as  when  cultures  killed  by  heat  are  used.  While 
the  results  of  these  investigations  fully  warrant  the  conclu- 
sions drawn  by  the  authors,  it  is  doubtful  if  the  method 
will  be  generally  approved  as  applicable  to  man.  The  pos- 
sibility of  accident  where  living  cultures  are  used  even 

Philippine  Journal  of  Science,  Section  B,  1907,  p.  155;    1912,  p.  223. 
2  Zeit.  f.  Hyg.  Infektionskr.,  1903,  S.  45. 


BACILLUS  PEST  IS  399 

though  they  be  attenuated  to  the  point  of  harmlessness, 
as  decided  by  animal  tests,  is  more  than  likely  to  operate 
against  the  routine  employment  of  such  cultures  in  the 
protection  of  human  beings  by  vaccination. 

Besredka,  of  the  Pasteur  Institute,1  advocates  the  use 
of  a  "sensitized  vaccine"  against  plague.  This  consists  of 
dead  pest  bacilli  (killed  by  heat)  that  have  been  mixed  with 
antiplague  immune  serum  obtained  from  an  artificially 
immunized  animal.  It  is  claimed  by  him  that  the  process 
of  sensitizing  lessens  the  toxic  action  of  the  dead  bacteria; 
diminishes  the  risk  run  by  injecting  them  and  eliminates 
the  uncomfortable  local  and  constitutional  reactions  that 
so  often  accompany  the  injections;  while  at  the  same  time 
the  protective  properties  of  the  "vaccine"  are  preserved. 
Rowland,2  in  a  critical  review  of  the  subject,  fails  to  find 
any  neutralization  of  the  toxic  properties  of  the  dead  bacteria 
through  sensitization,  but  states  that  Besredka's  "vaccine" 
possesses  good  immunizing  power  and  users  of  it  have 
reported  favorably  as  to  the  minimum  of  discomfort  fol- 
lowing its  inoculation.  The  principle  here  used  has  been 
applied  by  Besredka,  Gay  and  others  to  the  making  of  pro- 
tective agents  for  other  types  of  infection. 

Antiplague  Serum. — The  general  principles  that  are 
involved  in  the  induction  of  immunity  with  antibody 
formation  hold  for  plague  as  for  a  number  of  other  types 
of  infection;  that  is  to  say,  the  repeated  injection  into  sus- 
ceptible animals  of  non-fatal  doses  of  the  specific  organism 
or  the  products  of  its  growth  and  disintegration,  results  in  the 
elaboration  in  the  injected  animal  of  substances  that  are  in 
one  way  or  another  antidotal,  destructive  or  neutralizing  for 
the  matters  injecteoT. 

1  Bull,  de  1'Institute  Pasteur,  1910,  viii,  p.  241 ;    1912,  x,  p.  529. 

2  Journal  of  Hygiene,  Plague  Supplement  II,  1912,  p.  344. 


400     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

In  the  effort  to  secure  specific  antiplague  serum  two 
general  plans  have  been  followed:  one,  by  the  repeated 
injection  of  horses  with  at  first  increasing  doses  of  dead 
pest  bacilli  followed  by  ascending  doses  of  the  living  organ- 
ism (Yersin's  method);1  the  other  by  the  injection  of  the 
toxic  extractives  from  artificially  treated  plague  bacilli  (the 
method  of  Lustig2).  The  former  method  aims  to  establish 
an  antibacterial  immunity,  the  latter  an  antitoxic  immunity. 

By  both  modes  of  procedure  sera  are  obtained  that  possess 
some  degree  of  curative  value  in  the  treatment  of  plague, 
but  in  both  instances  this  is  low.  When  tested  on  human 
beings  sick  of  plague  under  as  well  controlled  conditions  as 
are  offered  by  a  good  hospital,  it  was  concluded  by  the 
Indian  Plague  Commission:3  "From  the  whole  inquiry 
therefore  it  appears  that  the  administration  of  the  available 
sera  is  not  a  practicable  means  of  bringing  about  any  material 
diminution  in  the  mortality  from  plague  in  India.  It  may 
well  be  that  better  results  would  be  obtained  if  the  treatment 
could  be  commenced  within  a  few  hours  of  the  onset  of 
the  disease,  this  however,  is  in  the  great  majority  of  cases, 
impossible  in  ordinary  practice." 

The  investigations  of  the  Pest  Commissions  of  Germany, 
Austria,  and  Egypt,  as  well  as  those  of  the  Institutes  for 
Infectious  Diseases  at  Berlin,  Berne,  and  the  Pasteur  Insti- 
tute of  Paris,4  have  contributed  much  additional  informa- 
tion of  importance  to  this  subject.  They  confirm  the  orig- 
inal views  upon  the  protective  or  prophylactic  value  of  the 
antiplague  serum,  but  demonstrate  that  as  a  therapeutic 
agent  it  is  of  but  limited  usefulness. 

1  Annales  de  1'Institute  Pasteur,  1897,  p.  81. 

2  Deutsche  med.  Wchnschr.,  1897,  No.  15. 

3  Journal  of  Hyg.,  Plague  Supplement  II,  Seventh  Report,  1912,  p,  326. 

4  The  important  literature  bearing  on  this  subject  is  appended  to  the 
report  of  Kolle,  Hetsch,  and  Otto  (Zeitschr.  f.  Hygiene,  Bd.  xlviii,  p.  368). 


CHAPTER  XX. 

Some  of  the  Pathogenic  Organisms  Encountered  in  the  Mouth  Cavity  in 
Health  and  Disease — Micrococcus  Lanceolatus,  Micrococcus  Tetragenous, 
Bacterium  Influenzas,  Bacillus  Tuberculosis,  etc. 

USUALLY  in  the  course  of  certain  diseases,  and  from  time 
to  time  in  health,  pathogenic  bacteria  are  to  be  found  in 
the  mouth.  In  the  latter  instance  the  organisms,  while 
often  fully  pathogenic,  as  shown  by  tests  on  animals,  do 
apparently  no  harm  to  their  hosts,  with  whom  they  live 
in  a  commensal  relationship.  Moreover,  they  are  often  not 
regularly  and  persistently  present — at  times  they  may 
disappear  permanently,  at  other  times  they  may  be  recur- 
rent, with  varying  intervals,  for  longer  and  shorter  periods. 
The  "pneumococcus,"  as  it  is  called;  the  Micrococcus 
tetragenous;  the  influenza  bacillus,  the  Bacillus  diphtherias, 
and  the  ordinary  pyogenic  streptococci  may  be  cited  as 
occasional  guests  in  the  normal  mouth  cavity.  In  diphtheria, 
tonsillitis,  influenza  and  tuberculosis,  the  specific  organisms 
of  these  diseases  may  usually  be  detected  either  in  the  ordi- 
nary saliva  or  in  the  sputum  brought  up  from  the  deeper 
respiratory  tract. 

To  familiarize  one's  self  with  these  organisms  and  the 
customary  technique  for  their  isolation  one  may  proceed  as 
follows : 

Obtain  from    a    tuberculous   patient  a  sample  of  fresh 

sputum — that  of  the  morning  is  preferable.     Spread  it  in 

a  thin  layer  upon  a  black  glass  plate  and  select  one  of  the 

small,  white,  cheesy  masses  or  dense  mucous  clumps  scat- 

26  (401) 


402     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

tered  through  it.  With  a  pointed  forceps  smear  this  carefully 
upon  two  or  three  thin  cover-slips,  dry  and  fix  them  in  the 
way  given  for  ordinary  cover-slip  preparations.  Stain  one 
with  Loffler's  alkaline  methylene-blue  solution,  another  by 
the  Gram  method,  and  a  third  after  the  method  given  for 
bacterium  tuberculosis  in  fluids  or  sputum. 

In  that  stained  with  Loffler's  blue — slip  No.  1 — will  be 
seen  a  great  variety  of  organisms — round  cells,  ovals,  short 
and  long  rods,  perhaps  spiral  forms.  But  not  infrequently 
will  be  seen  diplococci  having  more  or  less  of  a  lancet  shape, 
joined  together  by  their  broad  ends,  the  points  of  the  lancet 
being  away  from  the  point  of  juncture  of  the  two  cells. 
There  may  also  be  seen  masses  of  cocci  which  are  conspicuous 
by  their  arrangement  into  groups  of  fours,  the  adjacent 
surfaces  being  somewhat  flattened. 

In  the  slip  stained  by  the  Gram  method  the  same  groups 
of  cocci  which  grow  as  threes  and  fours  will  be  seen;  but 
the  lancet-shaped  diplococci  may  now  present  an  altered 
appearance — they  are  usually  surrounded  by  a  capsule. 
This  capsule  is  very  delicate  in  structure,  and,  though  a 
frequent  accompaniment,  is  not  constant.  It  can  sometimes 
be  demonstrated  by  the  ordinary  methods  of  staining, 
though  the  method  of  Gram  is  most  satisfactory.  (Fig.  75.) 

In  the  third  slip,  which  has  been  stained  by  the  method 
given  for  tubercle  bacteria  in  sputum,  if  decolorization  has 
been  properly  conducted  and  no  contrast-stain  has  been 
employed,  the  field  will  be  colorless  or  of  only  a  very  pale 
rose  color.  None  of  the  numerous  organisms  seen  in  the 
first  slip  can  now  be  detected;  but  instead  there  will  be 
seen  scattered  through  the  field  very  delicate,  stained  rods, 
which  present,  in  most  instances,  a  conspicuous  beading  of 
their  protoplasm — that  is,  the  staining  is  not  homogeneous, 


PATHOGENIC  ORGANISMS  IN  MOUTH  CAVITY    403 

but  at  tolerably  regular  intervals  along  each  rod  are  seen 
alternating  stained  and  unstained  points.  These  rods  may 
be  found  singly,  in  groups  of  twos  and  threes,  and  sometimes 
in  clumps  consisting  of  large  numbers.  When  in  twos  or 
threes  it  is  not  uncommon  to  find  them  describing  an  X  or  a 
V  in  their  mode  of  arrangment,  or  again  they  may  be  seen 
lying  parallel  the  one  to  the  other. 

If  contrast-stains  are  used,  these  rods  will  be  detected 
and  recognized  by  their  retaining  the  orginal  color  with 

FIG.  74 


v  vvv  it' 

'  -fif*      ^\  '&>': 

^  H  ^      il       C        '"'"  ' 
•"'  -A          /*  /  ^      t.'         // 


Tuberculous  sputum  stained  by  Gabbett's  method.    Tubercle  bacteria  seen 
as  red  rods;    all  else  is  stained  blue. 

which  they  had  been  stained;  whereas  all  other  bacteria 
in  the  preparation,  as  well  as  the  tissue-cells  which  are  in 
the  sputum,  will  take  up  the  contrast-color.  (Fig.  74.) 

This  delicate,  beaded  rod  is  bacterium  tuberculosis.  The 
lancet-shaped  diplococcus  with  the  capsule  is  bacterium  pneu- 
monia?. The  cocci  grouped  in  fours  are  sarcina  tetragena. 

Inoculation  Experiment. — Inoculate  into  the  subcutaneous 
tissues  of  a  guinea-pig  one .  of  the  small,  white,  caseous 
masses,  similar  to  that  which  has  been  examined  micro- 


404      APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

scopically.    If  death  ensue,  it  will,  in  all  probability,  be  the 
result  of  one  of  the  three  following  types  of  infection : 

a.  Septicemia  resulting  from  the  introduction  into  the 
tissues  of  bacterium  pneumonia. 

b.  A  less  active  form  of  septicemia  resulting  from  the 
introduction  of  sarcina  tetragena,  an  organism  frequently 
seen  in  the  sputum. 

c.  Local  or  general  tuberculosis. 

SPUTUM   SEPTICEMIA.      BACTERIUM   PNEUMONIAS 
(WEICHSELBAUM),  MIGULA,  1900. 

SYNONYMS:  Diplococcus  pneumoniae,  Weichselbaum,  1886;  Pneumo- 
coccus,  Frankel,  1886;  Micrococcus  of  sputum  septicemia;  Diplococcus 
lanceolatus;  Streptococcus  lanceolatus;  Streptococcus  pasteuri;  Micro- 
coccus  lanceolatus. 

If  at  the  end  of  twenty-four  to  thirty-six  hours  the  animal 
be  found  dead,  we  may  reasonably  predict  that  the  result 
was  produced  by  the  introduction  into  the  tissues  of  the 
organism  of  sputum  septicemia  above  mentioned,  viz., 
bacterium  pneumonia,  which  is  not  uncommonly  found  in 
the  mouths  of  healthy  individuals  as  well  as  in  other  con- 
ditions. 

Inspection  of  the  site  of  inoculation  usually  reveals  a 
local  reaction.  "  This  may  be  of  a  serous,  fibrinous,  hemor- 
rhagic,  necrotic,  or  purulent  character.  Frequently  we  may 
find  combinations  of  these  conditions,  such  as  fibrino-puru- 
lent,  fibrino-serous,  or  sero-hemorrhagic."1  The  most  con- 
spicuous naked-eye  change  undergone  by  the  internal  organs 
will  be  enlargement  of  the  spleen.  It  is  usually  swollen,  but 
may  at  times  be  normal  in  appearance.  It  is  sometimes 

1  Welch,  Johns  Hopkins  Hospital  Bulletin,  December,  1892,  vol.  iii, 
No.  27. 


SPUTUM  SEPTICEMIA  405 

hard,  dark  red,  and  dry;  or  it  may  be  soft  and  rich  in  blood. 
Frequently  there  is  a  limited  fibrinous  exudation  over  por- 
tions of  the  peritoneum. 

Except  in  the  exudations,  the  organisms  are  found  only 
in  the  lumen-  of  the  bloodvessels,  where  they  are  usually 
present  in  enormous  numbers.  In  the  blood  they  are  prac- 
tically always  free,  being  but  rarely  found  within  the  bodies 
of  leukocytes. 

FIG.  75 


Bacterium  pneumonias  in  blood  of  rabbit.     Stained  by  method  of  Gram 
Decolorization  not  complete. 


In  stained  preparations  from  the  blood  and  exudates  a 
capsule  is  not  infrequently  seen  surrounding  the  organisms. 
(Fig.  75.)  This,  however,  is  not  constant. 

If  a  drop  of  blood  from  the  dead  animal  be  introduced 
into  the  tissues  of  a  second  animal  (mouse  or  rabbit),  iden- 
tically the  same  conditions  will  be  reproduced. 

If  the  organism  be  isolated  in  pure  culture  from  the  blood 
of  the  animal,  and  a  portion  of  this  culture  be  introduced 
into  the  tissues  of  a  susceptible  animal,  we  shall  see  again 
the  same  pathological  picture. 


406      APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

It  must  be  remembered,  however,  that  this  organism  when 
cultivated  for  a  time  on  artificial  media  may  lose  rapidly 
its  pathogenic  properties.  If,  therefore,  failure  to  reproduce 
the  disease  after  inoculation  with  old  cultures  should  occur, 
it  is  in  all  probability  due  to  such  loss  of  virulence. 

This  organism  was  discovered  by  Sternberg  in  1880.  It 
was  subsequently  described  by  A.  Frankel  as  the  etiological 
factor  in  the  production  of  actue  fibrinous  pneumonia. 

It  is  not  uncommonly  present  in  the  saliva  of  healthy 
individuals,  having  been  found  by  Sternberg  in  the  oral 
cavities  of  about  20  per  cent,  of  healthy  persons  examined 
by  him,  and  certain  Authors  are  of  the  opinion  that  it  occurs 
in  the  oral  or  nasal  cavities  of  all  individuals  at  one  time 
or  another  during  life.  It  is  constantly  to  be  detected  in 
the  rusty  sputum  of  patients  suffering  from  acute  fibrinous 
pneumonia.  Its  presence  has  been  noted  in  the  middle  ear, 
in  the  pericardial  sac,  in  the  pleura,  and  in  the  serous  cavities 
of  the  brain;  and  indeed  it  may  penetrate  from  its  usual 
site  of  development  in  the  mouth  to  any  of  the  more  distant 
organs. 

The  organism  is  commonly  found  as  a  diplococcus,  though 
here  and  there  short  chains  of  four  to  six  individuals  may 
be  seen.  (Fig.  75.)  The  individual  cells  are  more  or  less 
oval,  or,  more  strictly  speaking,  lancet-shaped,  for  at  one 
end  they  are  commonly  pointed.  When  joined  in  pairs  the 
junction  is  always  at  the  broad  ends  of  the  ovals.  When 
in  chains  only  the  terminal  cells  are  pointed,  and  then  at 
their  distal  extremities. 

As  already  stated,  in  preparations  directly  from  the  sputum 
or  from  the  blood  of  animals  a  delicate  capsule  may  fre- 
quently be  seen  surrounding  them.  Though  fairly  constant 
in  preparations  directly  from  the  blood  of  animals  and  from 


SPUTUM  SEPTICEMIA  407 

the  sputum  or  lungs  of  pneumonic  patients,  the  capsule  is 
but  rarely  observed  in  artificial  cultures.  Occasionally  in 
cultures  on  blood-serum,  in  milk,  and  on  agar-agar  it  can, 
according  to  some  authors,  be  detected;  but  this  is  by  no 
means  constant,  or  even  frequent. 

Under  the  most  favorable  artificial  conditions  this  organism 
grows  but  slowly,  and  frequently  not  at  all. 

When  successfully  grown  upon  the  different  media  it 
presents  somewhat  the  following  appearances: 

On  gelatin  its  development  is  very  limited  and  often  no 
growth  at  all  occurs.  This  is  probably  due  in  part  to  the 
low  temperature  at  which  gelatin  cultures  must  be  kept. 
If  development  occurs,  the  growth  appears  as  minute  whitish 
or  blue-white  points  on  the  plates.  These  very  small  colonies 
are  round,  finely  granular,  sharply  circumscribed,  and 
slightly  elevated  above  the  surface.  They  do  not  cause 
liquefaction  of  the  gelatin. 

If  grown  in  slant-  or  stab-cultures,  the  surface  develop- 
ment is  very  limited;  along  the  needle-track  tiny  whitish 
or  bluish-white  granules  appear. 

On  nutrient  agar-agar  the  colonies  are  almost  transparent, 
more  or  less  glistening,  and  very  delicate  in  structure. 

On  blood-serum  development  is  more  marked,  though 
still  extremely  feeble,  appearing  as  a  cluster  of  isolated  fine 
points  growing  closely  side  by  side. 

Growth  on  potato  is  not  usually  observed. 

When  grown  in  milk  it  commonly  causes  an  acid  reaction 
with  coincident  coagulation  of  the  casein.  Some  varieties, 
especially  non-virulent  ones,  do  not  coagulate  milk.1 

It  is  not  motile. 

1  Welch,  loc.  cit. 


408     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

In  media  containing  inulin  acid  is  produced  as  a  result 
of  its  fermentive  action.  When  suspended  in  bile  or  in  a 
solution  of  bile  salts  the  organism  is  dissolved. 

(NOTE. — Compare  this  with  Streptococci  and  Staphylo- 
cocci.) 

It  grows  best  at  a  temperature  of  from  35°  to  38°  C.  Below 
24°  C.  there  is  usually  no  development,  but  in  a  few  cases 
it  has  been  seen  to  grow  at  as  low  a  temperature  as  18°  C. 
Above  42°  C.  development  is  checked. 

It  grows  as  well  without  as  with  oxygen.  It  is  therefore 
one  of  the  facultative  anaerobic  forms. 

Cultivation  of  this  organism  is  most  successful  when 
some  one  of  the  serum-agar  or  agar-gelatin  mixtures  is 
employed.  (See  the  medium.) 

It  may  be  stained  with  the  ordinary  aniline  staining 
reagents.  For  demonstrating  the  capsule  the  method  of 
Gram  and  the  acetic-acid  method  give  the  best  results. 
(See  Stainings.) 

This  organism  is  conspicuous  for  the  irregularity  of  its 
behavior  when  grown  under  artificial  conditions:  usually  it 
loses  its  pathogenic  properties  after  a  few  generations;  but 
again  this  peculiarity  may  be  retained  for  a  much  longer 
time.  Often  it  fails  to  grow  after  three  or  four  trans- 
plantations on  artificial  media,  though  at  times  it  may  be 
carried  through  many  generations. 

Inoculation  into  Animals. — The  results  of  inoculations  with 
pure  cultures  of  this  organism  are  also  conspicuous  for  their 
irregularity.  When  the  organism  is  of  full  virulence  the 
form  of  septicemia  above  described  is  usually  produced,  but 
at  times  it  is  found  to  be  totally  devoid  of  pathogenic  powers : 
between  these  extremes  cultures  may  be  obtained  possess- 


SPUTUM  SEPTICEMIA  409 

ing  every  variation  in  the  intensity  of  their  disease-produc- 
ing properties.  The  principal  pathological  conditions  that 
may  be  produced  by  the  inoculation  of  susceptible  animals 
with  this  organism  are,  according  to  the  degree  of  its  viru- 
lence, acute  septicemia,  spreading  inflammatory  exudations, 
and  circumscribed  abscesses.  All  three  of  these  conditions 
may  sometimes  be  produced  by  inoculating  rabbits  with  the 
same  cultures  in  varying  amounts. 

Rabbits,  mice,  guinea-pigs,  dogs,  rats,  cats,  and  sheep  are 
susceptible  to  infection  by  this  organism.  Chickens  and 
pigeons  are  insusceptible.  Young  animals,  as  a  rule,  are 
more  easily  infected  than  old  ones.  Rabbits  and  mice  are 
the  most  susceptible  of  the  animals  used  for  expermental 
purposes,  and  in  testing  the  virulence  of  a  culture  it  is 
well  to  inoculate  one  of  each,  for  the  same  culture  may 
sometimes  be  virulent  for  mice  and  not  for  rabbits,  or 
vice  versa. 

If  the  culture  is  virulent,  intravascular  or  intraperitoneal 
injections  into  rabbits  may  produce  rapid  and  fatal  sep- 
ticemia; while  subcutaneous  inoculation  of  the  same  material 
may  result  in  only  a  localized  inflammatory  process.  On 
the  other  hand,  subcutaneous  inoculation  of  less  virulent 
cultures  may  produce  a  local  process,  while  intravenous 
inoculation  may  be  without  result. 

This  organism  is  the  cause  of  a  number  of  pathological 
conditions  in  human  beings  that  are  not  usually  consid- 
ered as  related  to  one  another  etiologically.  It  is  always 
present  in  the  inflamed  area  of  the  lung  in  acute  fibrinous 
or  lobar  pneumonia;  it  is  known  to  cause  acute  cerebrospinal 
meningitis,  endo-  and  pericarditis,  certain  forms  of  pleuritis, 
arthritis  and  periarthritis,  and  otitis  media. 


410    APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

Varieties. — The  foregoing  general  description  of  pneumo- 
coccus  suffices  for  the  recognition  of  the  organism  as  it  is 
frequently  found  in  the  normal  upper  air  passages  and  in 
cases  of  pneumonia;  that  is,  it  is  a  lancet-shaped,  Gram- 
positive,  encapsulated  diplococcus,  having  the  property  of 
fermenting  inulin,  of  dissolving  in  bile  or  bile  salts  and  of 
usually  causing  septicemia  when  introduced  into  the  bodies 
of  mice  and  rabbits.  But  this  description  by  no  means 
includes  certain  other  important  aspects  of  the  subject 
that  have  been  revealed  by  special  researches. 

It  has  been  shown  that  the  variations  in  virulence  upon 
animals  of  those  pneumococci  isolated  from  the  mouth 
of  normal  human  beings  is  of  but  small  importance  to 
an  interpretation  of  the  role  of  the  organism  in  the  causa- 
tion of  pneumonia  in  man;  and  intimate  study  of  pneu- 
monias in  man,  together  with  the  organisms  associated  with 
them,  have  revealed  a  state  of  affairs  not  only  not  suspected 
a  few  years  ago,  but  of  the  utmost  importance  to  an  under- 
standing of  the  variations  in  the  disease;  of  the  greater 
fatality  of  one  expression  of  the  disease  over  another  and 
the  likelihood  of  the  transmission  -of  the  disease  from  the 
sick  to  the  well. 

These  studies  have  brought  out  the  fact  that  in  about  80 
per  cent,  of  all  cases  of  pneumonia  pneumococci  are  present 
that  are  markedly  different  in  their  specific  immunologic 
reactions  from  those  often  found  in  the  normal  mouth,  and 
that  are  distinguished  only  by  such  reactions.  Such  varie- 
ties of  pneumococcus  are  found  only  in  cases  of  pneumonia, 
and  if  more  than  one  case  of  pneumonia  occur  in  succession 
among  persons  domiciled  together  the  same  type  of  pneu- 
mococcus will  frequently  be  found  in  all  of  them;  suggesting 
the  transmission  of  this  particular  variety  of  the  disease 
from  one  person  to  another. 


PNEUMOCOCCUS,   VARIETIES  AND  VARIATIONS      411 

These  highly  pathogenic  types  of  pneumococcus  are 
rarely  found  in  the  normal  mouth,  except  in  case  of  persons 
in  close  contact  with  cases  of  pneumonia;  and  of  equal 
importance  is  the  fact  that  under  such  circumstances  the 
pneumococcus  found  in  the  mouth  of  the  normal  individual 
("the  contact")  is  identical  to  that  found  in  the  discharges 
from  the  lungs  of  the  particular  patient  suffering  from 
pneumonia  with  whom  he  has  been  in  contact.  Such 
virulent  pneumococci  ultimately  disappear  from  the  air 
passages  of  the  convalescent  from  pneumonia,  as  well  as 
from  the  mouth  of  the  healthy  contact. 

If  one  secure  from  the  normal  air  passages  the  pneumo- 
coccus commonly  found  there  and  at  the  same  time  secure  a 
culture  of  pneumococcus  from  a  case  of  typical  lobar  pneumo- 
nia, it  will  be  found  that  in  morphology  and  other  biological 
peculiarities  the  two  cultures  are,  as  a  rule,  indistinguishable 
the  one  from  the  other.  On  the  other  hand,  if  animals  be 
immunized  from  each  culture  it  will  be  found  that  the  blood 
serum  of  each  of  the  immune  animals  agglutinates  only  its 
homologous  cultures;  that  is  to  say,  the  serum  of  the  animal 
immunized  by  the  use  of  pneumococcus  from  the  case  of 
pneumonia  agglutinates  the  pneumococcus  from  only  that 
case  and  similar  cases,  but  not  the  pneumococcus  common 
to  the  normal  mouth  or  pharynx.  While  the  serum  from  the 
other  animal  agglutinates  only  the  pneumococcus  used  in 
the  immunization  of  that  animal.  In  other  words,  we  have 
specific  agglutinations. 

If  we  examine  in  the  same  manner  all  cases  of  pneumonia 
we  find  pneumococci  differing  specifically  in  their  agglu- 
tinating reactions  not  only  from  those  frequent  in  the 
normal  mouth,  but  with  various  manifestations  of  the 
disease  we  find  variations  in  the  virulent  pneumococci 
specifically  related  to  them. 


412     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

In  other  words,  certain  "  types "  of  pneumococcus  are 
most  common  to  this  than  to  that  expression  of  pneumonia 
and  are  more  or  less  identified  with  the  varying  fatalities 
of  the  disease. 

At  the  present  time,  four  types  of  pneumococci,  dis- 
tinguished from  one  another  by  specific  agglutinating 
reactions,  are  recognized  and  no  transmutation  from  one 
type  to  the  other  has  been  observed,  even  though  every 
experimental  effort  has  been  made  to  determine  if  such 
occurs. 

Types. — Type  I  pneumococcus  causes  between  30  and  50 
per  cent,  of  all  true  pneumonias  and  results  in  a  fatality  of 
almost  25  per  cent,  of  the  cases  with  which  it  is  associated. 

Type  II  pneumococcus  causes  from  about  15  to  33  per 
cent,  of  true  pneumonias  and  is  fatal  to  nearly  60  per  cent, 
of  the  cases  in  which  it  is  found. 

Type  III  pneumococcus  is  present  in  from  about  8  to  12 
per  cent,  of  lobar  pneumonias.  Its  presence  is  associated 
with  a  mortality  in  the  neighborhood  of  60  per  cent. 

This  type  (III)  is  distinguished  from  the  other  types  not 
only  by  its  specific  agglutinating  reactions,  but  by  the 
mucoid  character  of  its  growth  under  artificial  conditions 
and  its  tendency  to  develop  into  streptococcus-like  chains. 

Type  IV  pneumococcus  comprises  a  heterogeneous  group 
none  of  which  can  properly  be  included  in  either  of  the 
other  groups.  In  this  group  are  found  those  pneumococci 
so  often  present  in  the  saliva  of  normal  individuals  and 
which  were  regarded  at  one  time  as  the  specific  exciters  of 
pneumonia.  In  morphology  and  general  biological  par- 
ticulars the  organisms  in  this  group  are  alike,  but  by  the 
agglutinations  test  they  are  found  to  differ  from  one  another 
as  well  as  from  the  other  types.  Etiologically,  Type  IV 


PNEUMOCOCCUS—  TYPES  AND  TYPING  413 

pneumococci  are  of  less  importance  than  those  of  the  other 
groups.  They  are  associated  with  about  one-fifth  of  all 
pneumonias  and  cause  only  about  7  per  cent,  of  fatalities. 

Further,  when  by  appropriate  methods  of  procedure 
animals  have  been  immunized  from  these  groups,  the  blood 
serum  of  such  immune  animals  are  found  to  have  a  favor- 
able action  in  preventing  infection  in  normal  animals,  but 
here  too  there  is  a  specific  relationship,  for  the  serum  of 
animals  immune  from  either  Type  I,  II  or  III  is  impotent 
when  employed  against  infection  by  pneumococci  of  the 
types  not  used  for  immunization.  This  specific  relation- 
ship must  always  be  borne  in  mind  in  efforts  to  produce 
sera  possessing  either  prophylactic  or  curative  properties 
for  the  disease  pneumonia. 

By  an  interchange  of  result,  methods  and  materials 
between  various  laboratories  especially  identified  with  the 
development  of  these  ideas,  it  has  become  possible  to  stand- 
ardize the  "typing"  of  pneumococci  in  a  very  satisfactory 
manner.  Specific  antisera  from  animals  highly  immun- 
ized from  each  type  group  of  pneumococci  are  now  avail- 
able, and  by  the  correct  use  of  such  sera  in  performing  the 
agglutination  reaction,  we  may  easily  determine  to  which 
type  any  pneumococcus  in  question  belongs,  as  well  as 
form  an  approximate  estimate  as  to  the  probable  outcome 
of  the  case  of  pneumonia  from  which  that  pneumococcus 
was  obtained. 

In  consequence  of  all  this  we  are  obliged  to  modify  our 
views  formerly  held  on  the  relation  of  pneumococci  to 
pneumonia.  We  are  not  any  longer  justified  in  believing 
that  the  pneumococcus  found  in  the  normal  mouth,  under 
conditions  not  known  to  us,  changes  from  a  harmless  com- 
mensal to  a  dangerous  pathogenic  species.  All  modern 


414     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

trustworthy  evidence  is  to  the  contrary,  arid  we  now  believe 
that  with  pneumococci,  as  with  all  other  living  things,  there 
have  been  established  in  the  course  of  time,  as  a  result  of 
environmental  influences,  variations  of  a  type  species  result- 
ing in  the  acquisition  of  essentially  fixed  characters  of 
fundamental  importance. 

The  Mechanism  of  Pneumonic  Infection — The  most  impor- 
tant result  of  pneumococcus  infection  in  man  is  pneumonia. 
The  mechanism  of  the  origin,  course  and  recovery  from 
pneumonia  still  constitutes  o"ne  of  the  obscure  problems 
of  medicine,  even  though  special  investigations  have  shed 
much  light  upon  several  important  phases  of  the  subject. 

For  a  clear  appreciation  of  the  current  views  on  the 
essential  features  of  this  riddle,  we  must  bear  in  mind  several 
fundamental  facts: 

1.  That  pneumonia  is  not  invariably  the  consequence  of 
the  presence  of  pneumococci  upon  the  mucous  surfaces  or 
in  the  body,  for  that  organism  is  often  found,  fully  virulent 
for  experimental  animals  in  the  mouth,  nose  or  upper  air 
passages   of  persons   in   perfect   health.     ("Carriers"    and 
"Contacts.") 

2.  That  pneumonia,  when  not  terminating  fatally,  is  a 
self -limited  disease,  i.  e.,  the  signs  and  symptoms  increase 
from  the  start  until  a  point  is  reached,  "the  crisis,"  when 
their  severity  suddenly  begins  to  lessen  and  may  continue 
to  do  so  until  recovery  is  established. 

3.  That  up  to,  and  for  a  time  after  the  crisis,  often  far 
into  convalescence,  living  virulent  pneumococci  are  present 
in  the  lungs.    They  can  be  found  constantly  in  the  sputum 
and  often  in  smaller  or  larger  numbers  in  the  circulating 
blood.     Their  number  seems  at  times  to  be  affected  little, 
if  at  all,  by  the  forces  that  occasion  the  crisis. 


PNEUMONIA  415 

4.  That  the  pathogenic  activities  of  the  pneumococcus  are 
not  referable  to  an  extracellular  toxin,  properly  so  called, 
but  rather    to  an  endotoxic  component  that  is  liberated 
in  the  body  when  the  bacteria  are  disintegrated  and  that 
may  be  liberated  artificially  by  certain  solvents  and  under 
such  conditions  as  favor  autolysis,  i.  e.,    self-digestion  of 
the  bacteria. 

5.  That  in  the  blood  of  convalescents  from  pneumonia 
specific,  protective  antibodies  are  to  be  found,  but  as  they 
are  inconstant  both  as  to  their  presence  and  as  to  their 
amounts  it  is  impossible  to  decide  their  role  in  the  mechanism 
of  recovery. 

6.  That  animals  may  be  actively  immunized  from  pneu- 
mococcus infection  with  but  little  difficulty,  but  the  serum 
from  such  animals  is  not  always  of  value  in  either  preventing 
infection  in  other  animals  in  which  it  is  injected  or  of  miti- 
gating or  curing  infections  already  established  in  animals. 

It  is  only  by  keeping  in  mind  the  foregoing  facts  that  we 
are  able  to  appreciate  the  difficulties  surrounding  the  problem 
of  pneumonia  or  to  properly  estimate  the  value  of  certain 
important  experimental  results  having  a  bearing  upon  it; 
notwithstanding  the  light  already  thrown  on  the  subject 
by  the  discovery  of  various  types  of  the  causative  organism 
and  the  development  of  knowledge  upon  their  several  pecu- 
liarities. 

Given  a  group  of  persons  with  either  of  the  established 
types  of  pneumococci  in  their  mouths,  noses  and  pharynges, 
why  is  it  that  some  may  develop  pneumonia  and  others 
remain  in  health? 

It  has  been  customary  to  reply:  that  in  those  developing 
the  disease  there  has  been  a  lessening  of  the  general  vitality 


416      APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

(resistance)  through  a  variety  of  agencies,  to  a  point  that 
enables  the  pneumococcus,  hitherto  present  only  in  a  com- 
mensal relationship,  to  exhibit  its  pathogenic  activities. 
This  is  plausible,  but  that  is  all.  There  is  nothing  definite 
in  the  way  of  experimental  evidence  to  support  it. 

The  most  satisfying  explanation*  of  the  beginnings  of 
pneumonia  is  that  offered  by  the  investigations  of  Meltzer1 
and  his  associates.  They  demonstrated  that  if  fairly  large 
amounts  (5  or  6  c.c.)  of  fluid  cultures  of  pneumococci  be 
insufflated  into  the  lungs  of  dogs,  that  many  of  the  bron- 
chioles became  occluded  as  the  result  of  the  exudation 
following  such  insufflations.  The  occlusion  converts  the 
termini  of  those  bronchioles,  with  their  alveoli,  into  tiny 
cavities.  In  such  cavities  the  pneumococci  develop  and 
produce  irritating  substances  which  in  time  bring  about 
more  or  less  extensive  inflammation  of  the  lung  tissues 
round  about  them.  The  characteristics  of  these  inflamma- 
tory areas  are  in  all  important  details  identical  with  those 
of  true  pneumonia  in  man.  This  experimentally-produced 
pneumonia  is  not,  however,  clinically  identical  with  pneu- 
monia in  man,  as  it  is  not  accompanied  by  the  crisis,  nor 
does  one  observe  the  sequence  of  local  changes  leading  to 
resolution  that  are  commonly  noticed  in  the  course  of  pneur 
monia  in  man.  Nevertheless,  the  results  of  this  investiga- 
tion justify  the  conception  that  pneumonia  in  man  may 
not,  after  all,  be  from  the  start  a  matter  purely  and  simply 
of  the  invasion  of  the  lung  by  pneumococci,  but  rather  that 
for  such  invasion  to  be  followed  by  the  characteristic  lesions 
of  the  disease,  there  must  first  exist  physical  conditions 
favorable  to  the  massed  or  circumscribed  development  of 

Uour.  Exp.  Med.,  1912,  xv,  133. 


PNEUMONIA  417 

the  organism.  In  the  light  of  Meltzer's  studies  one  can 
conceive  that  through  one  or  another  of  many  causes 
exudations,  non-specific  in  character,  may  occur  in  the 
lungs,  occlude  terminal  bronchi  and,  as  in  the  experimental 
cases,  cause  small  cavities  into  which  pneumococci,  gaining 
access,  develop  as  in  a  closed  space — and  by  the  products 
of  their  growth  bring  about  progressive  inflammation  of 
the  tissues  surrounding  them.  The  experimental  evidence 
also  suggests  the  view  that  pneumonia  probably  always 
starts  as  such  isolated  patches  which,  by  extension,  coalesce 
until  finally  larger  areas  or  indeed  whole  lobes  of  the  lungs 
are  involved.  When  this  inflammation  of  the  lung,  with  its 
accompanying  symptoms,  have  progressed  for  about  a  week, 
the  crisis  may  be  expected,  i.  e.,  the  distressing  symptoms 
become  more  or  less  suddenly  relieved,  fever  begins  to 
decline,  respiration  is  less  difficult,  and  there  are  beginning 
signs  of  changes  in  the  diseased  lung  tissue,  i.  e.,  resolution 
may  set  in. 

These  sudden  changes  for  the  better,  so  often  observed 
in  true  lobar  pneumonia,  and  as  said,  denominated  "the 
crisis,"  constitute  one  of  the  dramatic  phenomena  of  clinical 
medicine.  As  if  by  magic,  often  within  a  few  hours,  a  patient 
apparently  in  extremis,  may  be  found  in  comparative  comfort 
and  progressing  steadily  to  recovery  with  little  or  no  return 
of  the  distressing  symptoms.  It  is  needless  to  say  that  this 
is  not  the  history  of  every  case,  but  it  is  so  frequently  seen 
in  non-fatal  cases  as  to  fairly  characterize  the  course  of  a 
case  destined  to  recover. 

What  are  the  forces  that  work  this  remarkable  change  for 

the  better?    It  cannot  be  that  the  pneumococci  causing  the 

trouble  are  suddenly  killed  off  and  their  hurtful  action  in 

this  way  terminated;  for  we  have  seen  that  long  after  the 

27 


418      APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

crisis  they  may  be  found  in  the  sputum  of  the  patient  alive, 
fully  virulent  and  in  almost  countless  numbers.  It  has  been 
suggested  that  after  about  a  week  there  develops  in  the 
tissues  of  the  body  a  sufficient  amount  of  antibodies  to 
neutralize  the  .poison  of  the  pneumococci  and  that  coincident 
with  this  neutralization  there  is  a  cessation  of  the  evil 
effects,  i.  e.,  the  crisis  occurs.  Vague  as  this  may  appear 
it  is  probably  as  satisfactory  as  any  other  explanation 
available  at  this  time.  There  are  objections  or  criticisms 
that  may,  however,  be  offered  in  discussing  it.  If  that  be 
the  correct  explanation  of  the  crisis,  one  might  reasonably 
expect  to  detect  in  the  blood  of  convalescents  from  pneumonia 
protective  antibodies  in  sufficient  amount  and  with  such 
constancy  as  to  support  the  view,  but  such  is  not  always 
the  case.  In  some  instances  antibodies  are  found  in  the 
blood  immediately  after  the  crisis  in  such  amounts  that  a 
fraction  of  1  cubic  centimeter  of  the  serum  will  protect 
a  mouse  from  infection  by  a  hundred  fold  the  ordinary 
fatal  dose  of  virulent  homologous  pneumococci;  in  other 
cases  no  such  protective  bodies  are  to  be  demonstrated  at 
all;  in  the  majority  of  cases  limited  amounts  of  such  pro- 
tective agents  are  to  be  demonstrated.  In  some  cases  pro- 
tective bodies  may  be  detected  in  the  blood  a  few  hours 
after  the  crisis,  and  none  may  be  found  a  few  days  later. 
It  is  such  inconstancies  as  these  that  call  into  question 
the  explanation  offered  above,  or  at  least  justify  the  sus- 
picion that  the  crisis  may  be  dependent  upon  other  forces 
in  addition  to  those  having  to  do  with  the  neutralization  of 
poison  or  the  destruction  of  a  certain  number  of  the  germs. 
It  has  been  suggested  that  such  other  factors  may  com- 
prise provisions  for  preventing  further  growth  of  the  pneu- 
mococci in  the  tissues  without  actually  killing  them  or 


PNE  UMONIA—IMM  UNIZA  TION  419 

robbing  them  of  their  power  to  produce  infection  when 
removed  alive  from  the  pneumonic  patient. 

It  also  has  been  suggested  that  the  crisis  constitutes  the 
advent  of  a  refractory  state  on  the  part  of  the  tissues— a 
state  having  some  analogies  to  anaphylactic  shock.  As 
yet  this  can  be  taken  only  as  a  suggestion.  Much  more  in 
the  way  of  experimental  evidence  is  needed  before  it  can 
be  accepted. 

It  is  scarcely  suitable  to  a  book  of  this  character  to  pursue 
all  the  lines  of  argument  that  have  been  advanced  in  con- 
nection with  this  subject.  It  suffices  to  say  that  at  present 
we  are  forced  still  to  speculate  as  to  the  nature  of  at  least 
some  of  the  important  factors  responsible  for  the  self  limi- 
tation of  this  desease. 

Immunization  and  Specific  Antisera. — Little  difficulty  has 
been  experienced  in  the  efforts  to  actively  immunize  animals 
from  pneumococcus  infection.  Horses  have  been  carried 
to  such  a  high  degree  of  immunization  by  repeated  intra- 
venous injection  of  pneumococcus  cultures  that  as  much  as 
2500  c.c.  of  a  virulent  culture  has  been  injected  into  the 
veins  at  one  time. 

From  such  highly  immunized  animals  sera  have  been 
obtained  of  remarkable  potency  in  preventing  infection; 
thus  Cole  found  that  0.2  c.c.  of  serum  from  one  of  his 
immunized  horses  would  protect  a  mouse  from  a  million- 
fold  the  lethal  dose  of  virulent  pneumococci,  provided  the 
serum  and  the  culture  be  injected  into  the  animal  at  the 
same  time.  But  if  the  animal  be  first  infected,  then  the 
serum  has  practically  no  saving  powers  even  though  it  be 
injected  only  a  few  hours  later  and  in  very  much  larger 
amounts;  in  fact,  Cole  states,  it  is  difficult  or  impossible 
to  rescue  the  animal,  no  matter  how  much  serum  is  injected. 


420     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

We  see  then  that  while  active  immunization  is  compara- 
tively easy  of  accomplishment,  the  matter  is  altogether 
different  when  the  serum  of  animals  so  immunized  is  used 
for  therapeutic  purposes.  The  failure  of  serum  from  im- 
munized animals  to  assist  in  the  cure  of  pneumonia  or 
other  pneumococcus  infection  with  certainty  is  variously 
explained,  but  as  yet  none  of  the  explanations  are  univer- 
sally accepted.  By  some  it  is  believed  that  immune  serum 
has  not  been  used  in  sufficient  quantities;  by  others  it  is 
believed  that  if  the  intensity  of  the  infection  exceeds  a 
certain  degree  that  no  amount  of  immune  serum  will  suffice 
to  rescue.  This  latter  view  is  particularly  applicable  to 
pneumonia,  a  disease  in  which  one  is  dealing  with  an  unusu- 
ally severe  type  of  infection  associated  with  enormous 
numbers  of  bacteria  in  the  body. 

Cole  suggests  that  the  failure  of  immune  serum  to  exhibit 
its  curative  powers  in  the  cure  of  pneumonia  may  not  be 
due  to  too  small  amounts  of  serum  used,  but  rather  to  an 
inability  on  the  part  of  the  infected  body  to  supply  the 
factors  necessary  to  complement  the  action  of  the  serum. 
His  investigations  lead  him  to  several  important  conclu- 
sions, among  which  may  be  mentioned:  Since  pneumococci 
may  be  divided  into  several  distinct  groups,  it  is  necessary 
to  use  for  curative  purposes  a  serum  from  an  animal  immu- 
nized from  a  strain  of  pneumococci  belonging  to  the  same 
group  as  that  with  which  the  patient  is  infected.  In  order 
to  be  effective  antipneumococcus  serum  must  be  adminis- 
tered early  and  in  large  doses.  With  these  facts  in  mind  the 
treatment  of  human  beings  suffering  from  pneumonia  with 
homologous,  immune  serum  has  resulted  in  very  low  mor- 
tality. In  cases  so  treated  the  bacteria  in  the  blood  are 
destroyed  and  specific  immune  substances  appear  in  the 


INFECTION  WITH  SARCINA  TETRAGENA    421 

blood  very  promptly  after  the  injection  of  the  serum.  A 
part  of  the  action  of  the  immune  serum  seems  to  be  anti- 
toxic.1 

INFECTION  WITH   SARCINA   TETRAGENA    (GAFFKY), 
MIGULA,    1900. 

SYNONYM:     Micrococcus  tetragenus,  Gaffky,  1883. 

Should  the  death  of  the  animal  not  occur  within  the  first 
twenty-eight  to. thirty  hours  after  inoculation,  but  be  post- 
poned until  between  the  fourth  and  eighth  day,  it  may 
result  from  the  invasion  of  the  tissues  by  the  organism  now 
to  be  described,  viz.,  sarcina  tetragena. 

This  organism  was  discovered  by  Gaffky,  and  was  subse- 
quently described  by  Koch  in  the  account  of  his  experiments 
upon  tuberculosis.  It  is  often  present  in  the  saliva  of 
healthy  individuals  and  is  commonly  present  in  the  sputum 
of  tuberculous  patients.  Koch  found  it  very  frequently  in 
the  pulmonary  cavities  of  phthisical  patients.  It,  however, 
plays  no  part  in  the  etiology  of  tuberculosis.  It  is  principally 
of  historic  interest,  being  of  little  pathogenic  significance. 

It  is  a  small  round  coccus  of  about  IM  transverse  diam- 
e£er.  It  is  seen  as  single  cells,  joined  in  pairs,  and  in 
threes;  but  its  most  conspicuous  grouping  is  in  fours,  from 
which  arrangement  it  takes  its  name.  In  preparations  made 
from  cultures  of  this  organism  it  is  not  rare  to  find  single 
bodies  which  are  much  larger  than  the  other  individuals  in 
the  field.  Close  inspection  reveals  them  to  be  cells  in  the 
initial  stage  of  division  into  twos  and  fours.  A  peculiarity 
of  this  organism  is  that  the  cells  are  bound  together  by  a 
transparent  gelatinous  mass. 

1  Cole,  Jour.  Am.  Med.  Assoc.,  1912,  lix,  693  and  1913,  xli,  663. 


422     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

When  cultivated  artificially  it  grows  very  slowly. 

Upon  gelatin  plates  the  colonies  appear  as  round,  sharply 
circumscribed,  punctiform  masses  which  are  slightly  elevated 
above  the  surface  of  the  surrounding  medium.  Under  a 
low  magnifying  power  they  are  seen  to  be  slightly  granular 
and  to  present  a  more  or  less  glassy  lustre. 

The  colonies  increase  but  little  in  size  after  the  third  or 
fourth  day.  If  cultivated  as  stab-cultures  in  gelatin,  there 
appears  upon  the  surface  at  the  point  of  inoculation  a  cir- 
cumscribed white  point,  slightly  elevated  above  the  surface 
and  limited  to  the  immediate  neighborhood  of  the  point 
of  inoculation.  Down  the  needle-track  the  growth  is  not 
continuous,  but  appears  in  isolated,  round,  dense  white 
clumps  or  beads,  which  do  not  develop  beyond  very  small 
points. 

It  does  not  liquefy  gelatin. 

Upon  plates  of  nutrient  agar-agar  the  colonies  appear  as 
small,  almost  transparent,  round  points,  which  have  about 
the  same  color  and  appearance  as  a  drop  of  egg-albumen; 
they  are  very  slightly  opaque.  They  are  moist  and  glisten- 
ing. They  rarely  develop  to  an  extent  exceeding  1  to  2 
mm.  in  diameter. 

Upon  agar-agar  as  stab-  or  slant-cultures  the  surface 
growth  has  more  or  less  of  a  mucoid  appearance.  It  is 
moist,  glistening,  and  irregularly  outlined.  The  outline  of 
the  growth  depends  upon  the  moisture  of  the  agar-agar. 
It  is  slightly  elevated  above  the  surface  of  the  medium. 

In  contradistinction  to  the  gelatin  stab-cultures,  the 
growth  in  agar-agar  is  continuous  along  the  track  of  the 
needle. 

The  growth  on  potato  is  a  thick,  irregular,  slimy-looking 
patch. 


INFECTION  WITH  SARCINA  TETRAGENA         423 

The  transparent  mucilaginous  substance  which  is  seen 
to  surround  these  organisms  renders  them  coherent,  so 
that  efforts  to  take  up  a  portion  of  a  colony  from  the  agar- 
agar  or  potato  cultures  result  usually  in  drawing  out  fine, 
silky  threads,  consisting  of  organisms  imbedded  in  the 
mucoid  material. 

The  organism  grows  best  at  from  35°  to  38°  C.,  but  can 
be  cultivated  at  the  ordinary  room-temperature — about 
20°  C. 

The  growth  under  all  conditions  is  slow. 

It  grows  both  in  the  presence  of  and  without  oxygen. 

It  is  not  motile. 

It  stains  readily  with  all  the  ordinary  aniline  dyes. 

In  tissues  its  presence  is  readily  demonstrated  by  the 
staining-method  of  Gram. 

The  grouping  into  fours  is  particularly  well  seen  in  sec- 
tions from  the  organs  of  animals  dead  of  this  form  of  septi- 
cemia.  In  such  sections  the  organisms  will  always  be  found 
within  the  capillaries. 

INOCULATION  INTO  ANIMALS. — To  the  naked  eye  no  altera- 
tion can  be  seen  in  the  organs  of  animals  that  have  died  as 
a  result  of  inoculation  with  sarcina  tetragena;  but  micro- 
scopic examination  of  cover-slip  preparations  from  the  blood 
and  viscera  reveals  the  presence  of  the  organisms  throughout 
the  body — especially  is  this  true  of  preparations  from  the 
spleen.  White  mice  and  guinea-pigs  are  susceptible  to  the 
disease.  Gray  mice,  dogs,  and  rabbits  are  not  susceptible 
to  this  form  of  septicemia.  Subsequent  inoculation  of 
healthy  animals  with  a  drop  of  blood,  a  bit  of  tissue,  or  a 
portion  of  a  pure  culture  of  this  organism  from  the  body  of 
an  animal  dead  of  this  disease,  results  in  a  reproduction  of 
the  conditions  found  in  the  dead  animal  from  which  the 
tissues  or  cultures  were  obtained. 


424      APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

It  sometimes  happens  that  in  guinea-pigs  which  have 
been  inoculated  with  this  organism  local  pus-formations 
result,  instead  of  a  general  septicemia.  The  organisms  will 
then  be  found  in  the  pus-cavity. 

BACTERIUM  INFLUENZA   (E.   PFEIFFER),  LEHMANN 
AND  NEUMANN,   1896. 

SYNONYM:    Influenza  bacillus,  R.  Pfeiffer,  1892. 

Influenza  is  one  of  the  important  historic  epidemic 
diseases,  on  the  nature  of  which  much  light  has  been  shed 
through  modern  methods  of  investigation.  Quoting  Hirsch: 
—the  first  trustworthy  literary  records  that  we  have  of 
this  disease  date  from  the  early  part  of  the  twelfth  century. 

Between  1173  and  1874  it  made  its  epidemic  or  pan- 
demic appearance  on  eighty-six  different  occasions.  Its 
first  recorded  appearance  in  this  country  was  in  Massa- 
chusetts in  1672;  since  that  time  there  have  been  twenty- 
two  visitations  of  influenza  in  the  United  States.  The  pan- 
demic of  1889-90,  to  that  date  the  most  severe  for  a  long 
time,  appears  to  have  originated  in  Central  Asia  and  to 
have  spread  pretty  much  over  the  entire  civilized  world;  that 
of  1918  also  seems  to  have  had  its  origin  somewhere  in  the 
Orient  and  to  have  spread  along  lines  of  traffic,  principally 
by  human  contact. 

The  advent  of  influenza  in  a  community  is  always  remark- 
able for  its  astonishing  rate  of  transmission  from  person  to 
person  and  its  dissemination  over  wide  areas. 

During  the  pandemic  of  1889-90  investigations  having 
for  their  object  the  discovery  of  its  cause,  resulted  in  demon- 
strating in  the  catarrhal  secretions  from  the  air  passages  a 


BACTERIUM  INFLUENZA  425 

microorganism  that  is  claimed  to  stand  in  causal  relation 
to  influenza. 

This  organism,  bacterium  influenzse,  as  it  is  called,  was 
discovered,  isolated,  cultivated  and  described  by  R.  Pfeiffer. 

By  appropriate  methods  of  staining  it  is  also  frequently 
possible  to  demonstrate  the  presence  of  bacterium  influenzse 
in  the  secretions  of  the  nose,  mouth,  and  throat  of  appar- 
ently healthy  persons,  as  well  as  in  those  from  persons 
suffering  from  such  diseases  as  diphtheria,  scarlet  fever, 
measles,  etc. 

It  appears  to  be  a  widely  distributed  organism.  These 
facts  are  of  importance  and  must  be  borne  in  mind  in  con- 
nection with  the  contention  that  Bacillus  influenzse  is  not 
the  cause  of  influenza,  but  is  only  an  early  invader  after  the 
disease  has  been  started  by  some  other,  as  yet  unknown, 
living  virus. 

Between  the  epidemic  of  1889-90  and  that  of  1918  opin- 
ion was  in  general  concordant  in  regarding  this  bacillus 
as  the  exciting  cause  of  influenza.  During  and  since  the 
outbreak  of  1918,  however,  there  has  been  a  divergence 
of  opinion  on  the  subject;  some  still  believing  in  the 
etiological  relationship  of  Pfeiffer's  bacillus  to  the  disease, 
others  regarding  it  as  only  a  secondary,  though  early,  invader 
after  the  disease  itself  has  been  started  by  some  other  living, 
transmissible  agent.  Just  what  that  agent  is  cannot  now  be 
said,  but  there  is  some  ground  for  believing  that  it  may  be 
one  of  the  so-called  filterable,  ultramicroscopic,  amorphous 
viruses. 

If  this  latter  view  should  ultimately  prove  to  be  correct, 
we  shall  still  have  not  only  Bacillus  influenzse,  but  pneu- 
mococci  and  streptococci,  as  very  early  invaders  in  practic- 
ally cases,  and  as  agents  with  which  we  must  reckon;  for 


426      APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

there  can  be  no  doubt  that  singly  or  together  they  are 
responsible  for  most  of  the  clinical  symptoms  and  patho- 
logical changes  by  which  influenza  is  characterized. 

Bacterium  influenzae  is  a  very  small,  slender,  non-spore- 
forming,  non-motile,  aerobic  bacillus,  occurring  singly  and 
in  pairs,  joined  end  to  end.  It  stains  with  watery  solutions 
of  the  ordinary  basic  aniline  dyes;  somewhat  better  with 

FIG.  76 


Bacterium  influenzae  in  sputum. 

alkaline  methylene-blue,  but  best  when  treated  for  five 
minutes  with  a  dilution  of  Ziehl's  carbol-fuchsin  in  water 
(the  color  of  the  solution  should  be  pale  red).  (Fig.  76.) 
It  is  decolorized  by  the  method  of  Gram. 

It  develops  only  at  temperatures  ranging  from  26°  to 
43°  C.  Its  optimum  temperature  for  growth  is  37°  C.  It 
possesses  the  peculiarity  of  developing  upon  only  those 
artificial  culture  media  to  which  blood  or  blood-coloring- 


BACTERIUM  INFLUENZA  427 

matter  has  been  added.  Its  cultivation  is  best  conducted 
and  its  development  most  satisfactorily  observed  by  the 
following  procedure :  over  the  surface  of  a  slanted  agar  tube 
or  over  agar-agar  solidified  in  a  Petri  dish  smear  a  small 
quantity  of  sterile  blood  (not  blood-serum).  A  bit  of  the 
mucus  from  the  sputum  of  the  influenza  patient  is  then 
taken  up  with  sterilized  forceps  or  on  a  sterilized  wire  loop, 
rinsed  in  sterile  bouillon  or  water  and  rubbed  over  the  sur- 
face of  the  prepared  agar-agar.  The  plate  or  tube  is  then 
placed  in  the  incubator  at  37°  to  38°  C.  If  influenza  bacilli 
be  present,  they  will  develop  as  minute,  transparent,  watery 
colonies  that  are  without  structure,  and  which  resemble 
somewhat  minute  drops  of  dew.  They  are  discrete  and 
show  little  or  no  tendency  to  coalesce. 

If  a  small  bit  of  mucus  be  rubbed  over  the  surface  of 
ordinary  nutrient  agar-agar,  no  such  colonies  develop. 
In  making  the  diagnosis  by  this  method  cultures  on  both 
agar-agar  containing  blood  (not  blood-serum)  and  agar- 
agar  containing  no  blood  should  always  be  made,  for  the 
reason  that  growth  of  these  peculiar  colonies  in  the  former 
and  no  such  growth  in  the  latter  are  evidence  that  one  is 
dealing  with  the  organism  under  consideration. 

The  organism  may  also  be  cultivated  in  bouillon  to  which 
blood  has  been  added,  if  kept  at  body-temperature.  The 
growth  appears  as  whitish  flakes.  Since  this  organism  is 
a  strict  ae'robe,  its  cultivation  can  only  be  conducted  on 
the  surface  of  the  medium  used— i.  e.,  where  it  has  freest 
access  to  oxygen.  It  is  therefore  inadvisable  to  prepare 
plates  in  the  usual  way.  When  its  cultivation  is  attempted 
in  bouillon  it  is  recommended,  in  order  to  favor  the  free 
diffusion  of  oxygen,  that  the  depth  of  fluid  be  very  shallow. 

Contrary  to  what  might  be  supposed,  bacterium  influenzse 


428     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

has  very  little  tenacity  of  life  outside  of  the  diseased  body. 
It  is  destroyed  in  from  two  to  three  hours  by  rapid  drying, 
and  in  from  eight  to  twenty-four  hours  when  dried  more 
slowly.  Cultures  retain  their  vitality  for  from  two  to  three 
weeks.  The  organism  dies  in  water  in  a  little  over  a  day. 
As  a  result  of  these  observations,  Pfeiffer  did  not  believe 
the  disease  to  be  disseminated  by  either  the  air  or  the  water, 
but  rather  by  direct  infection  from  the  catarrhal  secretions 
of  the  patients.  During  the  outbreak  of  1918  this  opinion 
received  additional  confirmation,  though  some  of  the  disease 
spread  among  army  troops  at  that  time  is  believed  to  have 
been  referable  to  dirty  eating  utensils,  infected  food  residue 
and  lack  of  facilities  for  or  care  in  the  proper  conduct  of 
kitchens  and  mess-rooms. 

This  organism  has  not  been  found  outside  of  the  human 
body.  In  the  influenza  patient  it  is  present  very  early, 
practically  with  the  advent  of  symptoms,  in  the  catarrhal 
secretions  from  the  upper  air  passages  and  lungs.  It  may 
be  demonstrated  microscopically  in  the  mucus  by  cover- 
slip  preparations  made  in  the  usual  way  and  stained  with 
diluted  carbol-fuchsin,  referred  to  above.  In  the  tissues 
it  may  be'  demonstrated  in  sections  stained  in  the  same 
solution.  In  the  sputum  the  bacteria  are  found  as  masses 
and  as  scattered  cells.  (See  Fig.  76.)  They  are  also  found 
within  the  bodies  of  leukocytes,  especially  in  the  later 
stages  of  the  disease  when  convalescence  has  set  in;  at 
this  time  they  appear  as  very  small,  irregular,  evidently 
degenerated  bacteria  within  white  blood  corpuscles.  They 
are  also  present  in  the  nasal  secretions. 

At  autopsies  it  is  advisable  to  cut  out  pieces  of  the  diseased 
tissue  about  the  size  of  a  pea  or  a  bean,  break  them  up  in 
a  small  quantity  of  sterile  water  or  bouillon,  and  make  the 


BACTERIUM  INFLUENZA  429 

cultures  from  this  infusion.  By  this  procedure  two  advan- 
tages are  gained:  first,  a  dilution  of  the  number  of  bacteria 
present;  and,  secondly,  the  tissue  furnishes  the  amount  of 
hemoglobin  necessary  for  the  growth  of  the  organism.  Under 
these  circumstances  it  is,  of  course,  not  necessary  to  make 
a  further  addition  of  blood  to  the  culture-medium. 

The  only  animal  that  has  been  found  susceptible  to 
inoculation  with  this  organism  is  the  monkey.  By  intra- 
tracheal  injection  Pfeiffer  succeeded  in  causing  a  toxic 
condition  that  proved  fatal.  He  does  not  regard  the  death 
of  the  animals  as  due  to  general  infection,  but  rather  to 
intoxication.  The  disease,  as  seen  in  man,  has  not  been 
reproduced  in  animals. 


CHAPTER  XXL 

Tuberculosis — Microscopic  Appearance  of  Miliary  Tubercles — Diffuse 
Caseation — Cavity-formation — Encapsulation  of  Tuberculous  Foci — 
Primary  Infection — Modes  of  Infection — The  Bacterium  Tuberculo- 
sis— Location  of  the  Bacilli  in  the  Tissues — Microscopic  Appearance 
of  Bacterium  Tuberculosis — Staining  Peculiarities — Organisms  with 
which  Bacterium  Tuberculosis  may  be  Confounded:  Bacterium 
Leprae;  Bacterium  Smegmatis — Acid-proof  Bacteria — Bacterium  Tu- 
berculosis Avium — Variations — Pseudotuberculosis — Susceptibility  of 
Animals — Tuberculin— Vaccination  Against  '  Tuberculosis — Actino- 
myces  Bovis — Actinomyces  Israeli,  Actinomyces  Madura,  Actino- 
myces  Farcinicus,  Actinomyces  Eppingeri,  Actinomyces  Pseudotuber- 
culosis. 


BACTERIUM  TUBERCULOSIS   (KOCH),  MIGULA, 
1900. 

SYNONYM:    Bacillus  tuberculosis,  Koch,  1882. 

LOCAL  OR  GENERAL  TUBERCULOSIS.— Should  the  animal 
succumb  to  neither  of  the  infections  just  described,  then 
its  death  from  tuberculosis  may  reasonably  be  expected. 

When  this  disease  is  in  progress  alterations  in  the  lym- 
phatic glands  nearest  the  site  of  inoculation  may  be  detected 
by  the  touch  in  from  two  to  four  weeks.  They  will  then  be 
found  enlarged.  Though  not  constant,  tumefaction  and 
subsequent  ulceration  at  the  point  of  inoculation  may  be 
observed.  Progressive  emaciation,  loss  of  appetite,  and 
difficulty  in  respiration  point  to  the  existence  of  the  general 
tuberculous  process.  Death  ensues  in  from  four  to  eight 
weeks  after  inoculation.  At  autopsy  either  general  or  local 
tuberculosis  may  be  found.  The  expressions  of  tuberculosis 
are  so  manifold  and  in  different  animals  vary  so  widely  the 
(430) 


BACTERIUM  TUBERCULOSIS  431 

one  from  the  other,  that  no  fixed  law  as  to  what  will  appear 
at  autopsy  can  a  priori  be  laid  down. 

The  guinea-pig,  which  is  best  suited  for  this  experiment 
because  its  susceptibility  to  tuberculosis  is  greater  and  more 
constant  than  that  of  other  animals  usually  found  in  the 
laboratory,  presents,  in  the  main,  changes  that  are  charac- 
terized by  coagulation-necrosis  and  caseation.  This  is 
particularly  the  case  when  the  infection  is  general — i.  e., 
when  the  process  is  of  the  acute  miliary  type;  then  the  tissues 
of  the  liver  and  spleen  present  the  most  favorable  field  for 
the  study  of  this  pathological-anatomical  alteration. 

In  general,  the  tubercular  lesions  can  be  divided  into 
those  of  strictly  focal  character — i.  e.,  the  miliary  and  the 
conglomerate  tubercles — and  those  which  are  more  diffuse. 
The  latter  lesions,  although  primarily  of  the  same  nature 
as  the  miliary  tubercles,  are  much  greater  in  extent  and  not 
so  sharply  circumscribed.  These  latter  lesions  play  a  more 
conspicuous  role  in  the  pathology  of  the  disease  than  do 
the  miliary  nodules,  although  it  is  the  miliary  nodules 
(tubercles)  that  give  to  the  disease  its  name. 

At  autopsy  the  pathological  manifestations  of  the  disease 
are  not  infrequently  seen  to  be  confined  to  the  seat  of  inocu- 
lation and  to  the  neighboring  lymphatic  glands.  These 
tissues  then  present  all  the  characteristics  of  the  tuberculous 
process  in  the  stage  of  cheesy  degeneration.  When  the 
disease  is  more  general  the  degree  of  its  extension  varies. 
Sometimes  the  small  gray  nodules — miliary  tubercles — are 
only  to  be  seen  with  the  naked  eye  in  the  tissues  of  the  liver 
and  spleen.  Again,  they  may  invade  the  lung,  and  frequently 
they  are  distributed  over  the  serous  membranes  of  the 
intestines,  the  lungs,  the  heart,  and  the  brain.  These  gray 
nodules,  as  seen  by  the  naked  eye,  vary  in  size  from  that  of 


432     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

a  pin-point  to  that  of  a  hempseed,  and,  as  a  rule,  are,  in 
this  stage,  the  result  of  the  fusion  of  two  or  more  still  smaller 
foci.  Though  the  two  terms  "miliary"  and  "conglomerate" 
are  employed  for  the  description  of  the  macroscopic  appear- 
ances of  these  nodules,  yet  it  is  very  rarely  that  any  condition 
other  than  that  due  to  the  fusion  of  several  of  these  minute 
foci  can  be  detected  by  the  naked  eye. 

The  miliary  tubercles  are  of  a  pale  gray  color,  with  a 
white  center,  are  slightly  elevated  above  the  surface  of  the 
tissue  in  which  they  are  located,  and,  as  stated,  vary  con- 
siderably in  dimensions,  usually  appearing  as  points  which 
range  in  size  from  that  of  a  pin-point  to  that  of  a  pin-head. 
They  are  not  only  located  upon  the  surface  of  the  organs, 
but  are  distributed  through  the  depths  of  the  tissues.  To 
the  touch  they  sometimes  present  nothing  characteristic, 
but  when  closely  packed  together  in  large  numbers  they 
usually  give  a  mealy  or  sandy  sensation  to  the  hand 
passed  over  them.  Stained  sections  of  miliary  tubercles 
present  a  distinctly  characteristic  appearance,  and  the  dis- 
ease may  be  recognized  by  these  histological  changes  alone, 
though  the  crucial  test  in  the  diagnosis  is  the  demonstra- 
tion of  tubercle  bacilli  within  the  nodules. 

Microscopic  Appearance  of  Miliary  Tubercles. — A  miliary 
tubercule  under  a  low  magnifying  power  of  the  microscope 
presents  somewhat  the  following  appearance:  there  is  a 
central  pale  area,  evidently  composed  of  necrotic  tissue 
because  of  its  incapacity  for  taking  up  the  nuclear  stains 
commonly  employed.  Scattered  through  this  necrotic  area 
may  be  seen  granular  masses  irregular  in  size  and  shape; 
they  take  up  the  stains  employed  and  are  evidently  frag- 
ments of  cell-nuclei  in  course  of  destruction.  Throughout 
the  necrotic  area  may  be  seen  irregular  lines,  bands,  or 


BACTERIUM   TUBERCULOSIS  433 

ridges,  the  remains  of  tissues  not  yet  completely  destroyed. 
Around  the  periphery  of  this  area  may  sometimes  be  noticed 
large  multinucleated  cells,  the  nuclei  of  which  are  arranged 
about  the  periphery  of  the  cell  or  grouped  irregularly  at 
its  poles.  The  arrangement  of  these  nuclei  as  observed 
in  sections  is  usually  oval,  or  somewhat  crescentic.  In 
tubercles  from  the  human  subject  these  large  "giant-cells," 
as  they  are  called,  are  quite  common.  They  are  much  less 
frequent  in  tubercular  tissues  from  lower  animals. 

Round  about  the  central  focus  of  necrosis  is  seen  a  more 
or  less  broad  zone  of  closely  packed  small  round  and  oval 
bodies,  which  stain  readily  but  not  homogeneously.  They 
vary  in  size  and  shape,  and  are  seen  to  be  imbedded  in  a 
delicate  network  of  fibrinous-looking  tissue.  This  fibrin- 
like  network  in  which  these  bodies  lie,  and  which  is  a 
common  accompaniment  of  giant-cell  formation,  is  in  part 
composed  of  fibrin,  but  is  in  the  main,  most  probably,  the 
remains  of  the  interstitial  fibrous  tissue  of  the  part.  This 
zone  of  which  we  are  speaking  is  the  zone  of  so-called  "  granu- 
lation-tissue/' and  consists  of  leukocytes,  granulation-cells, 
fibrin,  and  the  fibrous  remains  of  the  organ;  the  irregularly 
oval,  granular  bodies  which  take  up  the  stain  are  the  nuclei 
of  these  cells.  The  zone  of  granulation-tissue  surrounds 
the  whole  of  the  tuberculous  process,  and  at  its  periphery 
may  fade  gradually  into  the  healthy  surrounding  tissues  or 
be  sharply  outlined  or  may  fuse  with  a  similar  zone  sur- 
rounding another  tubercular  focus. 

Diffuse  Caseation. — The  diffuse  caseation,  as  said,  plays 
a  more  important  role  in  the  tuberculous  lesion,  both  in  the 
human  and  experimental  forms,  than  does  the  formation 
of  miliary  tubercles.  Here  a  large  area  of  tissue  undergoes 
the  same  process  of  necrosis  and  caseation  as  the  center  of 
28 


434      APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

the  miliary  tubercle.  In  certain  tissues,  notably  the  lungs 
and  lymphatics,  it  is  more  marked  than  in  others.  In 
rabbits,  particularly,  all  the  changes  in  the  lung  frequently 
come  under  this  head.  When  this  is  the  case  solid  masses 
are  found,  sometimes  as  large  as  a  pea,  or  involving  even 
an  entire  lobe  or  the  whole  lung  in  some  cases.  They  are 
opaque  and  of  a  whitish-yellow  color,  and  on  section  are 
peculiarly  dry  and  hard.  Entire  lymphatic  glands  may 
be  changed  in  this  way.  The  conditions  which  appear  to  be 
most  favorable  to  the  occurrence  of  this  widespread  casea- 
tion  of  the  tissues  are  the  simultaneous  deposition  of  masses 
of  tubercle  bacilli  in  them,  and  the  involvement  of  a  wide 
area  instead  of  a  single  isolated  point,  as  in  the  miliary 
tubercle.  Necrosis  is  so  rapid  that  time  does  not  suffice  for 
those  reactive  changes  to  take  place  in  the  tissues  which 
result  in  the  formation  of  the  outer  zone  of  the  miliary 
tubercle.  In  other  instances  the  entire  caseous  area  is  sur- 
rounded by  a  granulation-zone  similar  to  that  around  the 
caseous  center  of  the  miliary  tubercles.  It  is  of  special 
importance  to  recognize  the  etiological  connection  between 
this'  diffuse  caseation  and  the  tubercle  bacillus,  because 
until  its  nature  was  accurately  determined  caseous  pneu- 
monia of  the  lungs  formed  the  chief  obstacle  which  many 
encountered  in  recognizing  the  specific  infectiousness  of 
tuberculosis. 

Cavity  Formation. — The  production  of  cavities,  a  prominent 
feature  in  human  tuberculosis,  particularly  of  the  lungs, 
is  due  to  softening  of  the  necrotic,  caseous  masses  or  of 
aggregations  of  miliary  tubercles.  The  material  softens, 
is  expelled  by  way  of  the  bronchi,  and  a  cavity  results. 
In  the  wall  of  this  cavity  the  tuberculous  changes  still  pro- 
ceed, both  as  diffuse  caseation  and  formation  of  miliary 


BACTERIUM   TUBERCULOSIS  435 

tubercles.  The  whole  cavity  with  the  reactive  changes  in 
the  tissues  of  its  walls  may  be  properly  conceived  as  a  single 
gigantic  tubercle,  its  wall  forming  a  tissue  very  analogous 
to  the  outer  zone  of  the  single  tubercle,  the  cavity  itself 
corresponding  to  the  caseous  center. 

In  animals  used  for  experiment  cavity  formation  of  this 
sort  is  very  rare,  owing  to  the  greater  resistance  of  the 
caseous  tissue.  That  it  is,  however,  possible  to  produce 
in  rabbits  conditions  that  eventuate  in  pulmonary  cavities 
in  all  physical  respects  similar  to  those  seen  in  the  human 
being  has  been  beautifully  demonstrated  by  Prudden.  He 
showed  that  when  he  had  injected  fluid  cultures  of  strepto- 
coccus pyogenes  into  the  trachea  of  rabbits  already  affected 
with  tubercular  consolidation  of  the  lungs,  the  result  of  the 
mixed  infection  thus  brought  about  was  cavity  formation 
in  eight  out  of  nine  lungs  subjected  to  the  conditions  of  the 
experiment;  while  in  only  one  out  of  eleven  did  cavities 
form  under  the  influence  of  the  tubercle  bacillus  alone.1  The 
investigations  of  Ayer2  not  only  confirm  the  findings  of 
Prudden,  but  reveal  additional  facts  of  very  great  practical 
importance.  He  demonstrated  that  experimental  cavity 
formation  is  very  largely  dependent  upon  the  mass,  physi- 
cally speaking,  of  tubercle  bacilli  used  for  the  intratracheal 
injection;  that  uncomplicated  tubercular  infection  is  not 
usually  accompanied  by  fever,  but  that  if  there  be  engrafted 
upon  such  infection,  another  type  of  infection  (in  Ayer's 
Experiments,  Streptococcus  Infection)  that  fever  was  ob- 
served in  something  over  69  per  cent,  of  the  animals 'used 
in  his  investigations. 

1  Prudden,   Experimental   Phthisis  in   Rabbits,   with   the  Formation  of 
Cavities,   etc.,   Transactions  of  the  Association  of  American  Physicians, 
1894,  ix,  166. 

2  Journal  of  Medical  Research,  November  2,  1914,  xxx,  141. 


436     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

In  the  contents  and  in  the  walls  of  tubercular  cavities 
in  man  bacteria  other  than  bacillus  tuberculosis  are  found. 
It  is  to  the  influence  of  some  of  these,  as  we  have  seen,  that 
diseases  other  than  tuberculosis  may  sometimes  be  produced 
by  the  inoculation  of  animals  with  the  sputum  from  such 
cases;  and  it  is  also  to  the  absorption  of  their  toxic  products 
that  some  of  the  constitutional  manifestations,  particularly 
fever,  commonly  seen  in  cases  of  advanced  pulmonary  tuber- 
culosis are  attributed. 

Encapsulation  of  Tubercular  Foci. — It  not  uncommonly 
occurs  that  round  about  a  necrotic  tuberculous  focus  there 
is  formed  a  fibrous  capsule  which  may  completely  shut  off 
the  diseased  from  the  healthy  tissue  surrounding  it;  or  a 
tuberculous  focus  may,  through  the  resistance  of  the  tissue 
in  which  it  is  located,  be  more  or  less  completely  isolated. 
In  this  condition  the  diseased  foci  may  lie  dormant  for  a 
long  time  and  give  no  evidence  of  their  existence,  until 
they  are  made  to  break  through  their  envelopes  by  some 
disturbing  cause.  With  the  passage  of  the  bacilli  from  such 
a  focus  into  the  vascular  or  lymphatic  circulation  the  disease 
may  become  general. 

It  is  to  some  such  accident  as  this  that  the  sudden  ap- 
pearance of  general  tubercular  infection  in  subjects  supposed 
to  have  recovered  from  the  primary  local  manifestations 
may  often  be  attributed.  The  breaking-down  of  old  caseous 
lymphatic  glands  is  a  common  example  of  this  recurrence  of 
tuberculosis. 

Primary  Infection. — Primary  infection  occurs  through 
either  the  vascular  or  lymphatic  circulation.  Through 
these  channels  the  bacilli  gain  access  to  the  tissues  and 
become  lodged  in  the  finer  capillary  ramifications  or  in  the 
more  minute  lymph  spaces.  Here  they  find  conditions 


BACTERIUM  TUBERCULOSIS  437 

favorable  to  their  development,  and  in  the  course  of  their 
life-processes  produce  substances  of  a  chemical  nature 
which  serve  to  bring  about  characteristic  changes  in  the 
immediate  neighborhood.  In  the  beginning  the  fixed  cells, 
particularly  the  endothelial  cells  of  the  capillaries  and 
lymph  spaces,  are  stimulated  to  proliferation.  With  the 
onset  of  this  phenomenon,  evidence  of  other  cell  multipli- 
cation may  readily  be  detected  in  and  about  the  affected 
focus.  As  proliferation  continues  and  as  the  local  circulation 
becomes  more  and  more  impaired,  the  center  of  the  diseased 
area  gradually  assumes  a  condition  of  inactivity,  and  ulti- 
mately presents  all  the  characteristics  of  dead  and  dying 
tissue.  This  death  of  tissue  is  one  of  the  earliest,  the  most 
easily  recognized,  and  the  most  characteristic  results  of 
tubercular  infection,  and  may  usually  be  detected,  in  greater 
or  less  degree,  even  in  the  youngest  and  most  minute  tuber- 
cles. With  the  production  of  this  progressive  necrosis — for 
progressive  it  is,  as  it  proceeds  as  long  as  the  bacilli  live  and 
continue  to  produce  their  poisonous  products — there  is 
in  addition  a  reactive  change  in  the  surrounding  tissues, 
which  results  in  the  formation  of  a  granulation  zone  at  the 
outer  margins  of  the  dying  and  dead  tissue.  This  zone 
consists  of  small,  round  granulation  cells  and  of  leukocytes, 
all  of  wnich  are  seen  in  the  meshes  of  the  finer  fibrous  tissues 
of  the  part.  At  the  same  time  alterations  are  produced 
in  the  walls  of  the  vessels  of  the  locality;  these  tend  to 
occlude  them,  and  thus  the  process  of  tissue-death  is  favored 
by  a  diminution  of  the  amount  of  nutrition  brought  to  them. 
These  changes  may  continue  until  eventually  conglomerate 
tubercles,  widespread  caseation,  or  cavity  formation  results; 
or  from  one  cause  or  another  the  life-processes  of  the  bacilli 
may  be  checked  and  recovery  occur. 


438     APPLICATION  OF  METHODS  OF   BACTERIOLOGY 

Modes  of  Infection. — Experimentally,  tuberculosis  may 
be  produced  in  susceptible  animals  by  subcutaneous  inocu- 
lation, by  direct  injection  into  the  circulation,  by  injection 
into  the  peritoneal  cavity,  by  feeding  of  tuberculous  material, 
by  the  introduction  of  the  bacilli  into  the  air-passages,  and 
by  inoculation  into  the  anterior  chamber  of  the  eye. 

In  the  human  subject  the  most  common  portals  of  infec- 
tion are,  doubtless,  the  air-passages,  the  alimentary  tract, 
and  cutaneous  wounds.  When  introduced  subcutaneously 
the  resulting  process  finds  its  most  pronounced  expression 
in  the  lymphatic  system.  The  growing  bacilli  make  their 
way  into  the  lymphatic  spaces  of  the  loose  cellular  tissue, 
are  taken  up  in  the  lymph  stream  and  deposited  in  the 
neighboring  lymphatic  glands.  Here  they  may  remain  and 
give  rise  to  no  alteration  other  than  that  seen  in  the  glands 
themselves;  or  they  may  pass  on  to  neighboring  glands, 
and  eventually  be  disseminated  throughout  the  lymphatic 
system,  ultimately  reaching  the  vascular  system. 

Having  gained  access  to  the  bloodvessels  the  results  are 
the  same  as  those  following  intra vascular  injection  of  the 
bacilli,  namely,  general  tuberculosis  quickly  follows,  with 
the  production  of  miliary  tubercles  most  conspicuous  in 
the  lungs  and  kidneys;  less  numerous  in  the  spleen,  liver, 
and  bone-marrow. 

When  inhaled  into  the  lungs,  if  conditions  are  favorable, 
multiplication  of  the  bacilli  quickly  occurs.  Coincident 
with  their  growth  they  are  mechanically  pressed  into  the 
tissues  of  the  lungs.  As  multiplication  continues  some  are 
transported  from  the  primary  site  of  infection  to  healthy 
portions  of  the  lung-tissue,  where,  through  their  develop- 
ment, the  process  is  repeated. 

In  the  same  way  infection  by  way  of  the  alimentary  tract 


BACTERIUM  TUBERCULOSIS  439 

is  in  the  main  due  to  the  bacilli  being  forced  by  mechanical 
pressure  into  the  walls  of  the  intestines.  Investigation  has 
shown  that  lesions  of  the  intestinal  coats  are  not  necessary 
for  the  entrance  of  tubercle  bacilli  from  the  lumen  of  the 
gut  into  the  internal  organs  and  tissues.  They  may  be 
transported  from  the  intestinal  tract  into  the  lymphatics 
in  the  same  way  that  the  fat-droplets  of  the  chyle  find 
entrance  into  the  lymphatic  circulation. 

They  may  gain  access  to  the  tissues  by  way  of  the  tonsils. 

Unlike  most  pathogenic  organisms,  the  tubercle  bacillus 
is  resistant  to  drying.  When  thrown  off  from  the  lungs  in 
the  sputum  of  tuberculous  patients,  unless  special  precau- 
tions be  taken  to  prevent  it,  the  sputum  becomes  dried,  is 
ground  into  dust,  and  sets  free  in  the  atmosphere  the 
tubercle  bacilli  which  came  with  it  from  the  lungs,  and  which 
have  the  property  of  exciting  the  disease  in  susceptible 
persons  who  receive  them  into  the  nose  and  throat. 

Location  of  the  Bacilli  in  the  Tissues. — The  bacilli  will  be 
found  most  numerous  in  those  tissues  in  which  the  disease 
is  most  active. 

In  the  initial  stage  of  the  disease  the  bacilli  will  be  fewer 
in  number  than  later;  at  this  time  only  scattered  bacilli 
may  be  found;  later  they  are  more  numerous;  and,  finally, 
when  the  process  has  advanced  to  a  stage  easily  recognizable 
by  the  naked  eye,  ,they  are  distributed  through  the  granula- 
tion zones  in  clumps  and  scattered  about  in  large  numbers. 

In  the  central  necrotic  masses,  which  consist  of  cell- 
detritus,  it  is  rare  that  the  organisms  can  be  demonstrated 
microscopically.  It  is  at  the  periphery'  of  these  areas  and 
in  the  progressing  granular  zone  that  they  are  to  be  seen 
most  frequently. 


440     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

This  apparent  absence  of  the  bacilli  from  the  central 
necrotic  area  and  often  from  old  caseous  tissues  must  not 
be  taken,  however,  as  evidence  that  these  materials  are  not 
infective,  for  with  them  the  disease  can  be  reproduced  in 
susceptible  ,animals  by  inoculation.  A  conspicuous  example 
of  this  condition  is  seen  in  old  scrofulous  glands.  These 
glands  usually  present  a  slow  process,  are  commonly  caseous, 
and  always  possess  the  property  of  producing  the  disease 
when  introduced  into  the  tissues  of  susceptible  animals, 
but  yet  they  are  the  most  difficult  of  all  tissues  in  which 
to  demonstrate  microscopically  the  presence  of  tubercle 
bacilli. 

In  tubercles  containing  giant-cells  the  bacilli  can  usually 
be  demonstrated  in  the  granular  contents  of  these  cells. 
Frequently  they  will  be  found  accumulated  at  the  pole  of 
the  cell  opposite  to  that  occupied  by  the  nuclei,  as  if  there 
existed  an  antagonism  between  the  nuclei  and  the  bacilli. 
In  some  of  these  cells,  however,  the  distribution  of  the  bacilli 
is  seen  to  be  irregular,  and  they  will  be  found  scattered 
among  the  nuclei  as  well  as  in  the  necrotic  center  of  the 
cell.  As  the  number  of  bacilli  in  the  giant-cell  increases 
the  cell  itself  is  ultimately  destroyed. 

Tubercular  tissues  always  contain  the  bacilli  and  are 
always  capable  of  reproducing  the  disease  when  introduced 
into  the  body  of  a  susceptible  animal.  From  the  tissues 
of  this  animal  the  bacilli  may  be  obtained  and  cultivated 
artificially,  and  these  cultures  are  capable  of  again  produc- 
ing the  disease  when  further  inoculated.  Thus  are  fulfilled 
the  postulates  formulated  by  Koch  for  proving  the  etio- 
logical  role  of  an  organism  in  the  production  of  a  malady. 


BACTERIUM  TUBERCULOSIS  441 

BACTERIUM   TUBERCULOSIS. 

Of  the  three  pathogenic  organisms  liable  to  occur  in  the 
sputum  of  a  tuberculous  subject,  the  tubercle  bacillus  gives 
us  the  greatest  difficulty  in  our  efforts  at  cultivation. 

It  is  almost  an  obligate  parasite,  and  finds  c6nditions 
entirely  favorable  to  its  development  only  in  the  animal 
body.  On  ordinary  artificial  media  the  bacilli  taken  directly 
from  the  animal  body  grow  only  very  imperfectly,  or,  in 
many  cases,  not  at  all.  From  this  it  seems  probable  that 
there  is  a  difference  in  the  nature  of  individual  tubercle 
bacilli — some  appearing  to  be  capable  of  growth  in  the 
animal  tissues  only,  while  others  are  apparently  possessed 
of  the  power  to  lead  a  limited  saprophytic  existence.  It 
may  be,  therefore,  that  those  bacilli  which  we  obtain  as 
artificial  cultures  from  the  animal  body  are  offsprings  of 
the  more  saprophytic  varieties.  At  best,  one  never  sees 
with  the  tubercle  bacillus  a  saprophytic  condition  in  any 
degree  comparable  to  that  possessed  by  many  of  the  other 
organisms  with  which  we  have  to  deal. 

For  the  cultivation  of  bacillus  tuberculosis  directly  from 
the  tissues  of  the  animal,  the  best  method  is  that  recom- 
mended by  Koch,  viz.,  cultivation  upon  blood  serum.  Its 
parastitic  tendencies  are  so  pronounced  that  even  very 
slight  variations  in  the  conditions  under  which  one  endeavors 
to  isolate  bacillus  tuberculosis  from  the  tissues  may  cause 
total  failure.  It  is,  therefore,  necessary  that  the  injunc- 
tions for  obtaining  it  in  pure  culture  be  carefully  observed. 

Preparation  of  Cultures  from  Tissues. — Under  strict  aseptic 
precautions  remove  from  the  animal  a  diseased  organ — the 
liver,  spleen,  or  a  lymphatic  gland  being  preferable.  Place 
the  tissue  in  a  sterilized  Petri  dish,  and  dissect  out  with 


442     APPLICATION  OF  METHODS  OF   BACTERIOLOGY 

sterilized  scissors  and  forceps  the  small  tubercular  nodules. 
Place  each  nodule  upon  the  surface  of  the  blood  serum,  one 
nodule  in  each  tube,  and  without  attempting  to  break  it 
up  or  smear  it  over  the  surface,  leave  it  for  four  or  five  days 
in  the  incubator.  After  this  time  it  may  be  rubbed  over 
the  surface  of  the  serum.  The  object  of  this  is  to  give  to 
bacilli  in  the  nodule  an  opportunity  to  multiply,  under 
the  favorable  conditions  of  temperature  and  moisture, 
before  an  effort  is  made  to  distribute  them  over  the  surface 
of  the  medium.  It  is  best  to  dissect  away  twenty  to  thirty 
such  tubercles  and  treat  each  in  the  same  way.  Some  of 
the  tubes  will  remain  sterile,  others  may  be  contaminated 
by  extraneous  saprophytic  organisms  during  the  manipula- 
tion, while  a  few  may  give  the  result  desired,  viz.,  a  growth 
of  the  tubercle  bacillus  itself. 

The  blood  serum  upon  which  the  organism  is  to  be  cul- 
tivated should  be  comparatively  freshly  prepared — that  is, 
should  not  be  dry. 

After  inoculating  the  tubes  they  should  be  carefully 
sealed  to  prevent  evaporation  and  consequent  drying. 
This  is  done  by  burning  off  the  overhanging  cotton  plug 
in  a  gas-flame,  and  then  impregnating  the  upper  layers  of 
the  cotton  with  either  sealing-wax  or  paraffin  of  a  high 
melting-point;  or  by  inserting  over  the  burned  end  of  the 
cotton  plug  a  soft,  closely  fitting  cork  that  has  been  sterilized 
in  the  steam  sterilizer  just  before  using  (Ghriskey).  This 
precaution  is  necessary  because  under  the  most  favorable 
conditions  tubercle  bacilli  directly  from  the  animal  body 
show  no  evidence  of  growth  for  about  twelve  days  after 
inoculation  upon  blood  serum,  and,  as  they  must  be  re- 
tained during  this  time  at  the  body-temperature — 37.5°  C. — 
evaporation  would  take  place  very  rapidly  and  the  medium 
would  become  too  dry  for  their  development. 


BACTERIUM  TUBERCULOSIS  443 

If  these  primary  efforts  result  in  a  growth  of  the  bacilli, 
further  cultivations  may  be  made  by  taking  up  a  bit  of 
the  colony,  preferably  a  moderately  large  quantity,  and 
transferring  it  to  fresh  serum,  and  this  in  turn  is  sealed 
up  and  retained  at  body  temperature.  Once  having  ob- 
tained the  organism  in  pure  culture,  its  subsequent  culti- 
vation may  be  conducted  upon  the  glycerin-agar-agar 
mixture — ordinary  neutral  nutrient  agar-agar  to  which 
from  4  to  6  per  cent,  of  glycerin  has  been  added.  This  is 
a  very  favorable  medium  for  the  growth  of  this  organism 
after  it  has  accommodated  itself  to  its  saprophytic  mode 
of  existence,  though  blood  serum  is  perhaps  the  best  medium 
to  be  employed  in  obtaining  the  first  generation  of  the 
organism  from  tuberculous  tissues. 

The  organism  may  be  cultivated  also  on  neutral  milk 
to  which  1  per  cent,  of  agar-agar  has  been  added,  also  upon 
the  surface  of  potato,  and  likewise  in  meat-infusion  bouillon 
containing  from  4  to  6  per  cent,  of  glycerin. 

Cultures  of  the  tubercle  bacillus  are  characteristic  in 
appearance — once  having  seen  them  there  is  little  proba- 
bility of  subsequent  mistake.  They  appear  as  dry  masses, 
which  may  develop  upon  the  surface  of  the  medium  either 
as  flat  scales  or  as  coarse,  heaped  up,  granular  nesses. 
They  are  never  moist,  and  frequently  have  the  appearance 
of  dry  meal  spread  upon  the  surface  of  the  medium.  In 
the  lower  part  of  the  tube  in  which  they  are  growing — i.  e., 
that  part  occupied  by  a  few  drops  of  fluid  which  has  in  part 
been  squeezed  from  the  medium  during  the  process  of  solidi- 
fication, and  is  in  part  water  of  condensation — the  colonies 
may  be  seen  to  float  as  a  thin  pellicle  upon  the  surface  of  the 
fluid. 

The  individuals  composing  the  growth  adhere  so  tena- 


444      APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

ciously  together  that  it  is  with  the  greatest  difficulty  they 
can  be  separated.  In  even  the  oldest  and  dryest  cultures 
pulverization  is  impossible.  The  masses  can  only  be  sepa- 
rated and  broken  v  up  by  grinding  in  a  mortar  with  the 
addition  of  some  foreign  substance,  such  as  very  fine, 
sterilized  sand,  or  ground  glass,  etc. 

The  cultures  are  of  a  dirty-drab  or  brownish-gray  color 
when  seen  on  serum  or  glycerin-agar-agar. 

On  potato  they  grow  in  practically  the  same  way,  though 
the  development  is  much  more  limited.  On  this  medium 
they  are  of  nearly  the  same  color  as  the  potato  on  which 
they  are  growing.  When  cultivated  for  a  time  on  potato 
they  are  said  to  lose  their  pathogenic  properties. 

On  milk-agar-agar  they,  are  of  so  nearly  the  same  color 
as  the  medium  that,  unless  they  are  growing  as  character- 
istic mealy-looking  masses,  considerably  elevated  above  the 
surface,  their  presence  is  less  conspicuous  than  when  on 
other  media. 

In  bouillon  they  grow  as  a  thin  pellicle  on  the  surface. 
This  may  fall  to  the  bottom  of  the  fluid  and  continue  to 
develop,  its  place  on  the  surface  being  taken  by  a  second 
pellicle. 

The,  tubercle  bacillus  does  not  develop  on  gelatin  because 
of  the  low  temperature  at  which  this  medium  must  be  used. 

Microscopic  Appearance  of  Bacterium  Tuberculosis. — Micro- 
scopically the  organism  itself  is  a  delicate  rod,  usually 
somewhat  beaded  in  its  structure,  though  rarely  it  is  seen 
to  be  homogeneous.  It  is  either  quite  straight,  or  somewhat 
curved  or  bent  on  its  long  axis.  In  some  preparations 
involution-forms,  consisting  of  rods  a  little  clubbed  at  one 
extremity  or  slightly  bulging  at  different  points,  may  be 
detected.  Branching  forms  of  this  organism  have  been 


BACTERIUM  TUBERCULOSIS  445 

described.  It  varies  in  length — sometimes  being  seen  in 
very  short  segments,  again  much  longer,  though  never  as 
long  threads.  Usually  its  length  varies  from  2  to  5ju.  It 
is  commonly  described  as  being  in  length  about  one-fourth 
to  one-half  the  diameter  of  a  red  blood  corpuscle.  It  is 
very  slender.  (See  Fig.  74.) 

These  rods  usually  present,  as  has  been  said,  an  appear- 
ance of  alternate  stained  and  colorless  portions.  At  times 
these  colorless  portions  are  seen  to  bulge  slightly  beyond 
the  contour  of  the  rod,  and  in  this  way  give  to  the  rods  the 
beaded  appearance  so  commonly  ascribed  to  them.  These 
oval  colorless  areas  were  at  one  time  thought  to  be  spores. 
A  number  of  competent  observers  have  expressed  the  opinion 
that  the  rods  which  we  see  in  tubercular  lesions  and  which 
we  call  bacillus  tuberculosis  are  not,  strictly  speaking, 
bacilli,  but  are  fragments  or  developmental  phases  of  a  more 
highly  organized  fungus — possibly  related  to  the  strepto- 
thrices  or  actinomyces.  The  point  cannot  now  be  decided. 

Staining  Peculiarities. — A  peculiarity  of  this  organism  is 
its  behavior  toward  staining  reagents,  and  by  this  means 
alone  it  may  easily  be  recognized.  The  tubercle  bacillus 
does  not  stain  by  the  ordinary  methods.  It  possesses  a 
peculiarity  in  its  composition  that  renders  it  proof  against 
the  simpler  staining  processes.  It  is  therefore  necessary 
that  more  energetic  and  penetrating  reagents  than  the 
ordinary  watery  solutions  be  employed.  Experience  has 
taught  us  that  certain  substances  not  only  increase  the 
solubility  of  the  aniline  dyes,  but  their  penetration  as  well. 
Two  of  these  are  aniline  oil  and  carbolic  acid.  They  are 
employed  in  the  solutions  to  about  the  point  of  saturation. 
(For  the  methods  of  staining  B.  tuberculosis  see  Chapter 
on  Staining.) 


446      APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

Under  the  influence  of  heat  these  solutions  are  seen  to 
stain  all  bacteria  very  intensely — the  tubercle  bacilli  as 
well  as  other  forms.  If  we  subject  our  preparation,  which 
may  contain  a  mixture  of  tubercle  bacilli  and  other  species, 
to  the  action  of  decolorizing  agents,  another  peculiarity 
of  the  tubercle  bacillus  will  be  observed.  While  all  other 
organisms  in  the  preparation  give  up  their  color  and  become 
invisible,  the  tubercle  bacillus  retains  it  with- marked  tenacity. 
It  stains  with  great  difficulty;  but  once  stained  it  retains 
the  color  even  under  the  action  of  strong  decolorizing  agents. 

Variations  of  B.  Tuberculosis. — Theobald  Smith1  called 
attention  to  certain  very  conspicuous  differences  that  may 
be  observed  between  the  bacilli  obtained  from  human  and 
those  from  bovine  tuberculosis;  and  in  a  series  of  inocula- 
tion experiments  Ravenel  has  shown  that  for  a  large  number 
of  animal  species  tubercle  bacilli  of  bovine  origin  were  con- 
stantly more  virulent  than  .those  from  human  sources ;  both 
of  which  observations  have  been  repeatedly  confirmed. 

Susceptibility  of  Animals  to  Tuberculosis. — The  animals 
that  are  known  to  be  susceptible  to  tuberculosis  are  man, 
apes,  cattle,  horses,  sheep,  hogs,  guinea-pigs,  pigeons,  rab- 
bits, cats,  and  field-mice.  White  mice,  dogs,  and  rats 
possess  immunity  from  the  disease. 

Tuberculin. — The  filtered  sterile  products  of  growth  from 
old  fluid  cultures  of  the  tubercle  bacillus  represent  what  is 
known  as  tuberculin — a  solution  containing  a  group  of 
protein  substances  possessing  most  interesting  properties. 
When  injected  subcutaneously  into  healthy  subjects  tuber- 
culin has  no  effect;  but  when  introduced  into  the  body  of 
a  tuberculous  person  or  animal  a  pronounced  systemic 

1  Transactions  of  the  Association  of  American  Physicians,  1896,  xi,  275. 


BACTERIUM  TUBERCULOSIS  447 

reaction  results,  consisting  of  sudden  but  temporary  ele- 
vation of  temperature,  with,  at  the  same  time,  the  occur- 
rence of  marked  hyperemia  about  the  tuberculous  focus, 
a  change  histologically  analogous  to  that  seen  in  the  pri- 
mary stages  of  acute  inflammation.  This  zone  of  hyperemia, 
with  the  coincident  exudation  and  infiltration  of  cellular 
elements,  probably  aids  in  the  isolation  or  casting  off  of 
the  tuberculous  nodule,  the  inflammatory  zone  forming, 
so  to  speak,  a  line  of  demarcation  between  the  diseased  and 
healthy  tissue. 

As  a  curative  agent  for  tuberculosis,  tuberculin  has  not 
proved  worthy  of  the  confidence  that  was  at  first  accorded 
to  it.  Its  field  of  usefulness  is  now  almost  limited  to  the 
diagnosis  of  obscure  cases. 

In  veterinary  medicine  it  has  proved  trustworthy  as  a 
diagnostic  aid,  and  is  practically  everywhere  in  use  for  the 
detection  of  incipient  tuberculosis  in  cattle. 

VACCINATION  AGAINST  TUBERCULOSIS. — Experiments  by 
Pearson  and  Gilliland,  v.  Behring,  Calmette,  and  others  have 
shown  that  it  is  possible  to  partly  immunize  animals  with 
lowly  virulent  tubercle  bacteria  of  human  origin.  After  one 
or  two  injections  with  such  organisms  the  animals  showed  for 
a  time  some  degree  of  tolerance  to  the  more  highly  virulent 
bovine  strains.  The  results  of  experiments  in  this  direction 
have  been  so  encouraging  as  to  justify  further  research 
in  this  direction,  but  complete  immunity  has  not  as  yet 
resulted. 

We  have  reviewed  the  three  common  pathogenic  organ- 
isms that  may  be  encountered  in  the  sputum  of  tuberculous 
individuals.  Occasionally  other  species  may  be  present. 
The  pyogenic  forms  are  not  rarely  found,  and  for  some  time 


448     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

after  an  attack  of  diphtheria  the  bacillus  of  Loffler  is  demon- 
strable in  the  pharynx,  so  that  it,  too,  may  be  present  under 
exceptional  circumstances. 

Organisms  with  which  Bacterium  Tuberculosis  may  be  Con- 
fused.— It  is  important  to  note  that  in  the  study  of  tubercu- 
losis one  may  fall  into  error  unless  it  be  borne  in  mind  that 
there  is  a  group  of  microorganisms  whose  members  are  in 
many  respects  so  like  the  genuine  bacillus  tuberculosis  as 
easily  to  be  mistaken  for  it.  While  its  peculiar  micro- 
chemical  reaction  is  usually  sufficient  for  identification,  par- 
ticularly in  connection  with  human  pathological  lesions,  it  is 
well  to  remember  that  the  confusing  organisms  are  not  only 
characterized  by  the  same  staining  peculiarities  as  bacillus 
tuberculosis,  but  may  readily  be  mistaken  for  it  on  morpho- 
logical grounds  also.  Furthermore,  while  not  all  the  mem- 
bers of  this  group  are  capable  of  causing  disease,  some  of 
them  are  pathogenic  for  the  same  animals  that  are  suscep- 
tible to  true  tubercular  infection;  and  there  may  produce  in 
those  animals  lesions  which  are  distinguishable  from  genuine 
tubercles  only  by  their  finer  histological  structure.  A  few 
words  concerning  some  of  these  varieties,  with  a  brief 
summary  of  their  more  important  peculiarities,  may  not 
be  out  of  place. 

BACTERIUM  LEPILE. — Between  1879  and  1881  there  was 
described  by  Hansen  and  by  Neisser  an  organism,  a  bacillus, 
that  was  constantly  to  be  found  in  the  nodules,  characteristic 
of  leprosy.  For  this  organism  the  name  bacillus  leprce  was 
suggested.  Though  very  like  bacterium  tuberculosis  in 
both  morphology  and  staining  properties,  it  is,  however, 
a  little  shorter,  thicker,  and  much  less  homogeneously 
stained.  Its  presence  in  the  tissues  and  secretions  is  demon- 
strated by  the  same  method  as  that  employed  for  detecting 


BACTERIUM  TUBERCULOSIS  449 

bacillus  tuberculosis.  In  secretions  of  leprous  nodules, 
stained  by  the  ordinary  Koch-Ehrlich  process,  the  bacilli, 
crowded  together  in  the  large  so-called  "lepra  cells/1  are 
always  to  be  seen  in  great  abundance.  Numerous  efforts 
to  cultivate  bacillus  leprse  from  the  diseased  tissues  and  to 
reproduce  the  disease  fby  inoculation  have  led  to  little  more 
than  a  mass  of  confusing  results.  It  is  possible  that  a  recent 
observation  of  Johnston1  may  assist  in  clearing  away  some 
at  least  of  the  confusion.  Johnston  believes  the  acid-proof, 
so-called  bacilli,  seen  in  the  lepra  cells  to  be  developmental 
phases  of  a  streptothrix  which  is  itself  not  acid  proof.  His 
opinion  appears  to  be  justified  by  the  results  of  carefully 
made  culture  and  inoculation  studies.2 

BACTERIUM  SMEGMATTS. — In  1885  Alvarez  and  Tavel 
discovered  in  the  fatty  secretions  about  the  genitalia  an 
organism  that  suggested  the  bacterium  of  tuberculosis. 
Their  observation  has  been  abundantly  confirmed  by  others, 
and  the  organism  to  which  they  directed  attention  is  now 
regarded  as  pretty  commonly  present  in  the  smegma.  It 
is  known,  therefore,  as  the  smegma  bacterium  (bacterium 
smegmatis).  In  this  secretion  it  is  found  in  clumps  located 
upon  or  within  epithelial  cells.  It  stains  by  the  method 
used  in  staining  bacterium  tuberculosis.  It  has  no  patho- 
genic power.  It  is  said  to  have  been  artificially  cultivated 
upon  coagulated  hydrocele  fluid  and  in  milk. 

THE  ACID-PROOF  BACTERIA. — In  addition  to  the  species 
mentioned,  quite  a  group  of  other  "acid-proof"  bacteria, 
as  they  are  called,  have  been  described  by  different  inves- 
tigators. They  are  characterised  by  staining,  as  does  bac- 

1  Philippine  Journal  of  Science,  June,  1914,  vol.  ix,  No.  3,  Section  B, 
Tropical  Medicine,  p.  227. 

2  For  a  general  discussion  on  this  ubject,  together  with  literary  references 
see  Wolbach  and  Honeij,  Journal  of  Medical  Research,  1914,  xxix,  367. 

29 


450     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

terium  tuberculosis,  by  retaining  the  stain  to  a  greater  or 
less  extent  when  treated  with  decolorizers,  and  by  being 
in  many  instances  strikingly  like  bacterium  tuberculosis 
in  their  morphology.  The  members  of  this  group  seem 
to  be  distributed  pretty  widely  in  nature.  They  have  been 
detected  in  non-tuberculous  sputum,  in  gangrene  of  the 
lung,  in  the  normal  intestinal  contents  of  man  and  domes- 
tic animals,  in  certain  of  the  cold-blooded  species,  in  the 
soil,  in  fodder — i.  e.,  grass,  hay  and  seed — in  manure, 
and  in  butter.  They  are  not  regularly  found  under  any 
of  these  conditions,  and  they  are  rarely  present  in  very 
large  numbers.  Inasmuch  as  they  are  occasionally  en- 
countered under  circumstances  that  might  lead  one  to 
look  for  true  tubercle  bacilli,  and  since  they  possess  certain 
peculiarities  similar  to  those  by  which  it  has  been  the  cus- 
tom to  identify  bacillus  tuberculosis — i.  e.,  retention  of  the 
stain  when  acted  upon  by  acids  or  alcohol,  and  a  more 
or  less  delicate,  beaded  form — the  possibility  of  their  being 
confounded  with  that  organism  is  obvious.  In  consequence 
they  received  a  great  deal  of  attention  for  a  time. 

Space  does  not  permit  of  a  description  of  the  twenty 
odd  species  (?)  that  have  been  described  by  different  in- 
vestigators. It  will  suffice  to  say,  from  personal  study  of 
the  group,  that  in  all  probability  not  more  than  three,  per- 
haps only  two,  species  are  really  represented,  and  that  the 
remainder  may  fairly  be  regarded  as  varieties.  As  said, 
the  characteristic  common  to  all  the  members  of  this  group 
is  that  they  are  to  greater  or  less  extent  acid-proof — i.  e., 
when  once  stained  by  the  Koch-Ehrlich  or  Ziehl  process 
the  color  is  not  in  all  cases  removed  by  the  ordinary  acid 
decolorizers.  In  this  particular,  however,  there  is  such  a 
striking  difference  between  the  degree  of  their  resistance  to 


BACTERIUM  TUBERCULOSIS  451 

acid  decolorizers  and  that  of  the  tubercle  bacillus  as  to 
render  this  an  important  differential  aid;  for  instance,  the 
tubercle  bacillus,  when  stained,  may  be  treated  for  several 
minutes  with  so  strong  a  decolorizer  as  30  per  cent,  nitric 
acid  without  losing  its  color;  whereas,  none  of  the  members 
of  this  group  retain  their  color  after  a  few  seconds  of  such 
treatment,  and  particularly  if  it  be  followed  by  washing 
in  alcohol.  In  morphology  some  of  them  might  readily  be 
mistaken  for  bacillus  tuberculosis,  though  even  these  are 
usually  a  trifle  larger  and  less  delicately  formed  than  that 
organism;  others  are  at  once  differentiated  from  normal 
tubercle  bacilli,  but  have  somewhat  their  appearance  when 
degenerated  or  involuted;  still  others  have  nothing  in  their 
general  appearance  to  lead  to  confusion. 

When  mixed  with  other  bacteria,  as  is  the  case  in  the  soil, 
in  manure,  in  intestinal  contents,  etc.,  their  isolation  in 
pure  culture  is  a  matter  of  difficulty,  and  this  is  by  no  means 
lessened  by  the  fact  that  under  these  circumstances  they 
are  always  numerically  in  the  minority.  When  present 
in  butter,  their  isolation  offers  fewer  difficulties,  for  by  the 
injection  of  the  butter  containing  them  into  the  peritoneal 
cavity  of  guinea-pigs  conditions  are  created  that  favor  their 
development,  and  from  animals  so  treated  they  may  usually 
be  recovered  in  pure  culture. 

When  studied  in  pure  culture,  all  of  them  are  at  once 
distinguished  from  bacillus  tuberculosis  by  the  following 
group  characteristics:  they  are  of  relatively  rapid  growth, 
there  being  usually  an  abundant  development  on  glycerin- 
agar-agar  after  twenty-four  to  forty-eight  hours  at  body- 
temperature;  they  grow  well  but  less  rapidly  at  ordinary 
room-temperature — i.  e.,  at  18°  to  20°  C.;  they  grow  well 
in  litmus-milk,  and,  as  a  rule,  produce  alkali  that  causes 


452     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

the  color  to  become  a  deep  blue;  the  growth  on  agar-agar 
is  dry,  shrivelled,  and  wrinkled  in  appearance,  and  of  a  soft, 
mealy  consistency  in  some  cases  (Holler's  grass  bacillus 
II,  Rabinowitsch's  butter  bacillus,  for  instance),  while  in 
others  it  is  more  membranous,  as  in  the  case  of  Holler's 
timothy  bacillus.  We  have  never  seen  in  these  cultures 
the  hard,  coarse  granules  so  common  to  cultures  of  bacillus 
tuberculosis;  on  glycerin-agar-agar  some  of  them,  namely, 
the  timothy  bacillus  of  Holler  and  its  varieties,  grow  with 
a  distinct  orange  color,  while  others,  the  grass  bacillus  II 
of  Holler,  the  butter  bacillus  of  Rabinowitsch,  and  their 
closely  allied  varieties,  begin  as  a  grayish-white  deposit 
which  may  ultimately  become  of  a  pale  or  even  distinct 
salmon  color. 

When  pure  cultures  of  them  are  injected  into  such  animals 
as  rabbits  or  guinea-pigs,  some  of  them  have  no  effect,  and 
others  cause  lesions  of  more  or  less  importance,  the  result 
being  dependent  upon  the  quantity  employed  and  the  mode 
of  inoculation.  By  subcutaneous  or  intraperitoneal  injec- 
tion of  pure  cultures  the  result  is  usually  negative.  Occa- 
sionally the  superficial  lymphatic  glands  in  the  neighborhood 
of  the  site  of  inoculation  may  be  inflamed  and  purulent. 
This  we  have  seen  only  after  subcutaneous  inoculation. 
If  pure  cultures  be  injected  into  the  peritoneal  cavity  along 
with  some  sterile,  irritating  substance,  such  as  sterilized 
butter,  a  widespread  fibrinopurulent  peritonitis  is  commonly 
the  result. 

When  injected  directly  into  the  circulation  of  rabbits, 
the  kidneys  are  almost  uniformly  affected,  and  in  the  ma- 
jority of  instances  they  are,  singularly  enough,  the  only 
organs  in  which  lesions  are  to  be  detected.  If,  for  instance, 


BACTERIUM  TUBERCULOSIS  453 

a  cubic  centimeter  of  a  carefully  prepared  suspension  in 
bouillon  of,  let  us  say,  Holler's  grass  bacillus  II,  be  injected 
into  the  circulation  of  a  rabbit,  and  the  animal  be  killed 
after  twelve  to  fourteen  days,  the  kidneys  will  be  found 
marked  by  gray  or  yellowish  points  that  range  in  size  from 
that  of  a  pin-point  to  that  of  a  pin-head.  They  are  some- 
times very  few  in  number,  but  in  other  cases  both  kidneys 
may  be  thickly  studded  with  them.  Often  they  are  not 
elevated  above  the  cortex  of  the  organ,  but  in  as  many  cases 
they  are  sharply  defined,  yellow  in  color,  and  stand  up 
prominently  from  the  cortical  surface,  being  at  the  same 
time  so  adherent  to  the  capsule  that  the  removal  of  the 
latter  tears  them  out  bodily  from  the  substance  of  the  organ. 
In  the  very  early  stages  of  development  these  nodules  are 
often  difficult  to  distinguish  from  young  tubercles,  the  reac- 
tion of  the  tissues  being,  as  in  the  case  of  tubercles,  charac- 
terized by  proliferation  of  the  fixed  cells  with  little  evidence 
of  leukocytic  invasion;  later  on,  true  giant-cell  formation 
is  recorded  by  some  observers.  We  have  not  seen  this. 
Clumps  of  endothelial  nuclei  or  of  lymphoid  cells  that 
remotely  suggest  the  arrangement  seen  in  giant  cells  are 
often  encountered,  but  we  have  not  regarded  them  as  true 
giant  cells.  When  fully  developed,  the  nodule  may  present 
a  mixed  condition  of  caseation  and  suppuration.  The 
conditions,  as  a  whole,  when  advanced  suggest  a  low  grade 
of  inflammatory  reaction.  Occasionally  nodules  are  en- 
countered, especially  in  the  kidney,  that  cannot  be  dis- 
tinguished from  tubercles.  The  bacilli  are  always  to  be 
found  within  the  nodules;  most  frequently  as  single  rods  or 
clumps  of  rods,  occasionally  as  rosette-like  mycelia  very 
suggestive  of  the  characteristic  growth  of  the  actinomyces 


454     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

fungus  in  the  tissues.  We  have  also  observed  this  mode 
of  development  by  Bacillus  tuberculosis.  (Figs.  77  and  78.) 

It  is  important  to  note  the  difference  between  the  results 
of  intravenous  inoculation  of  rabbits  with  bacillus  tuber- 
culosis arid  with  the  organisms  under  consideration.  When 
bacillus  tuberculosis  is  employed,  the  lungs,  as  well  as  the 
kidneys,  are  always  involved,  while  with  the  grass  bacillus 
II,  the  timothy  bacillus,  and  the  butter  bacillus,  involve- 
ment of  the  lungs,  in  our  experiments,  has  been  the  exception 
rather  than  the  rule. 

Another  point  of  interest  is  the  lack  of  tendency  on  the 
part  of  the  non-tuberculous  process  to  progress  or  become 
disseminated. 

That  the  members  of  this  group  are  botanically  related 
to  bacillus  tuberculosis  there  seems  little  room  for  doubt; 
but  from  personal  study  and  from  available  evidence  from 
other  sources  it  appears  unlikely  that  they  are,  except 
experimentally,  concerned  in  disease  production  or  that  they 
are  of  importance  to  either  human  or  animal  pathology.1 

In  the  microscopic  examination,  particularly  of  urine, 
of  secretions  from  about  the  anus,  rectum,  and  genitalia, 
and  of  butter,  it  is  manifestly  of  importance  to  bear  in  mind 
the  existence  of  this  confusing  group,  for  it  is  in  such  secre- 
tions and  substances  that  its  members  are  most  often  en- 
countered. The  smegma  bacillus  and  the  butter  bacillus 
are  especially  liable  to  lead  one  into  error  of  diagnosis. 
This  is  less  apt  to  be  the  case  with  the  comparatively  rare 
lepra  bacillus. 

1  For  the  literature  on  "acid-proof"  bacilli,  see  Cowie,  Journal  of  Experi- 
mental Medicine,  1900,  v,  205. 


Fig.  77 


\  , 


'<3f 


Q  '   S'9       '"' 

I    *Jf,   *   & 


Showing  Aetinomyees  Development  of  Bacillus  Tuber- 
culosis in  Lung  of  Rabbit,  thirty  days  after  intravenous 
injection  of  suspension  of  the  organism,  o  , 


Fig.  78 


Showing  Aetinomyees  Development  of  Acid-resisting 
Bacteria  (Butter  Bacillus  of  Rabinowitsch)  in  Kidney  of 
Rabbit,  following  upon  intravenous  injection  of  suspension 
of  the  organism. 


BACTERIUM  TUBERCULOSIS  AVIUM  455 

BACTERIUM   TUBERCULOSIS   AVIUM   (MAFFUCCI), 
MIGULA,  1900. 

SYNONYMS:  Bacillus  tuberculosis  avium,  Maffucci,  1891;  Mycobacter- 
ium  tuberculosis  avium,  Lehmann  and  Neumann,  1896. 

From  time  to  time  fowls  are  known  to  suffer  from  a  form 
of  tuberculosis  that  in  a  number  of  ways  suggests  human 
or  mammalian  tuberculosis.  The  bacillus  causing  the  disease, 
the  so-called  bacillus  of  fowl  tuberculosis,  bacillus  tuber- 
culosis avium,  while  simulating  the  genuine  bacillus  tuber- 
culosis morphologically,  differs  from  it  both  in  cultural 
and  pathogenic  peculiarities.  Thus,  for  instance,  it  develops 
into  much  longer  and  somewhat  thinner  threads;  grows 
rapidly  on  media  without  glycerin  or  glucose;  does  not 
grow  on  potato;  develops  as  well  at  from  42°  to  43°  C.  as 
at  37°  to  38°  C.;1  its  virulence  is  not  diminished  by  cul- 
tivation at  43°  C.;  development  on  artificial  media  begins 
in  from  six  to  eight  days  after  inoculation;  young  cultures 
on  solid  media  are  whitish,  soft,  and  moist,  becoming  yel- 
lowish and  slimy  with  age;  it  is  somewhat  more  resistant 
to  drying  and  high  temperatures  than  the  bacillus  of  mam- 
malian tuberculosis;  the  results  of  its  pathogenic  activities 
are  almost  always  chronic,  are  rarely  located  in  the  lungs 
or  intestines,  but  are  especially  frequent  in  the  liver  and 
spleen;  the  lesions  are  conspicuously  rich  in  bacteria,  do 
not  show  the  central  necrotic  area  that  characterize  the 
mammalian  tubercle;  the  disease  is  transmissible  from  the 
hen  to  the  embryo  chick;  the  only  susceptible  mammal  is 
the  rabbit;  the  guinea-pig  and  dog  are  naturally  immune; 
it  has  the  same  micro-chemical  staining  reactions  as  mam- 

1The  normal  body-temperature  of  fowls  ranges  between  41.5°  and 
42.5°  C. 


456     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

malian  bacillus  tuberculosis;  it  has  never  been  certainly 
detected  in  human  tuberculosis. 

Some  are  inclined  to  regard  this  organism  as  but  a  variety 
of  genuine  bacillus  tuberculosis,  and  it  is  not  unreasonable 
to  believe  that  the  sojourn  of  that  organism  in  the  body  of 
a  refractory  animal,  whose  normal  temperature  is  so  high  as 
that  of  the  fowl,  when  not  fatal  to  the  organism,  might 
result  in  striking  modifications  of  certain  of  its  biological 
functions.  In  fact,  Nocard1  has  shown  that  if  the  genuine 
bacillus  tuberculosis  from  man  be  left  in  the  peritoneal  cavity 
of  chickens  (by  the  collodion-sac  method  of  Metchnikoff, 
Roux,  and  Sallembini,  which  see)  for  from  five  to  eight 
months,  they  will,  by  the  end  of  this  time,  have  become  so 
modified  in  their  biological  peculiarities  as  to  simulate  very 
closely  the  bacillus  of  fowl  tuberculosis. 

Moore2  reports  studies  on  bacterium  tuberculosis  avium 
in  an  epidemic  occurring  in  California.  He  obtained  pure 
cultures  by  inoculating  glycerin-agar  or  blood  serum  tubes 
directly  from  tuberculous  livers  and  spleens.  In  the  origi- 
nal cultures  little  difficulty  was  experienced  in  cultivating 
the  organism  on  glycerin-agar,  fresh  dog  serum,  Dorset's 
egg-medium,  potato,  and  glycerin-bouillon.  The  general 
cultural  peculiarities  observed  agreed  with  those  described 
by  Maffucci,  Nocard,  Straus  and  Gamaleia,  and  others. 
He  states  that  the  avian  tubercle  bacteria  as  found  in  the 
tissues  of  the  fowl  resemble  quite  closely  those  of  the  bovine 
and  human  varieties  in  their  size  and  general  morphology. 
The  average  length  of  a  large  number  of  measurements  was 
2.7  microns.  Moore  also  tested  the  pathogenesis  of  the 
freshly  isolated  avian  tubercle  bacteria  on  fowls,  rabbits, 

1  Annales  de  1'Institut  Pasteur,  1898,  p.  561. 
8  Journal  of  Medical  Research,  1904,  vol.  vi. 


ACTINOMYCETES  457 

guinea-pigs,  and  pigeons.  The  results  of  these  inocula- 
tions, however,  were  unsatisfactory,  as  were  also  feeding 
experiments  of  healthy  fowls  with  human  tuberculous 
sputum  rich  in  bacteria. 

Pseudotuberculosis. — Anatomical  lesions  very  suggestive 
of,  though  not  identical  with,  those  produced  by  bacillus 
tuberculosis,  have  also  from  time  to  time  been  observed  in 
mice,  rats,  guinea-pigs,  rabbits,  cats,  goats,  bovines,  hogs, 
and  man.  They  do  not  appear  to  be  of  a  specific  nature  as 
regards  etiology,  for  the  reason  that  different  authors  have 
described  different  organisms  as  the  causative  agents. 
These  affections  are  usually  classed  under  the  name  pseudo- 
tuberculosis. 

ACTINOMYCETES. 

The  term  actinomycetes  is  restricted  to  a  group  of  organ- 
isms having  morphological  affinities  with  the  bacteria  on 
the  one  hand  and  the  hyphomycetes  on  the  other.  They 
resemble  the  bacteria  in  that  they  occur  as  homogeneous 
threads  which  under  artificial  cultivation  may  become 
segmented  into  short  bacillus-  or  coccus-like  fragments. 
Furthermore,  they  are  unlike  the  molds  in  that  they  have 
not  a  double  wall;  are  not  filled -with  fluid  containing  gran- 
ules, and  the  segments  are  not  separated  from  one  another 
by  a  distinct  partition.  They  simulate  the  molds  in  that 
they  develop  from  spores  into  dichotomously  branching 
threads,  which  ultimately  form  colonies  having  more  or 
less  resemblance  to  true  mycelia.  Certain  of  the  threads 
composing  such  a  mycelium  become  fruit  hyphse,  breaking 
up  into  round,  glistening,  spore-like  bodies.  As  a  rule, 
these  spores  are  devoid  of  the  high  resistance  to  heat  exhib- 


458     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

ited  by  bacterial  spores,  and  are  stainable  by  the  ordinary 
methods. 

The  limits  of  this  group  are  ill  defined  and  its  recognized 
components  are  not  as  a  whole  well  understood. 

The  longest  known  and  most  carefully  studied  actinomy- 
cetes  are  act.  bovis,  act.  madurce,  act.  farcinicus,  and  act. 
Eppingeri,  although  many  other  varieties  have  been  en- 
countered in  association  with  important  and  interesting 
pathological  lesions. 

The  fact  that  certain  bacteria,  viz.,  B.  tuberculosis,  B. 
mallei,  B.  diphtherise  are,  as  a  rule,  segmented  and  occa- 
sionally show  a  tendency  to  branch,  has  led  to  their  being 
classified  at  times  with  the  actinomycetes.  On  this  point, 
however,  there  is  as  yet  no  concensus  of  opinion. 

It  is  interesting  to  note  that  the  pathological  lesions  in 
which  actinomycetes  have  been  detected  show  in  many  cases 
certain  similarities  to  true  tubercular  processes,  and  in  few 
instances,  save  for  the  absence  of  tubercle  bacteria,  as  we 
usually  see  them,  were  indistinguishable  from  tuberculosis. 

More  or  less  imperfectly  studied  varieties  of  actino- 
mycetes have  been  encountered  in  abscess  of  the  brain, 
cerebrospinal  meningitis,  endocarditis,  bronchopneumonia, 
pleuropneumonia,  pustular  exanthemata,  abscess  of  the  lung, 
bronchiectasis,  pulmonary  gangrene,  necrosis  of  the  vertebrae, 
subphrenic  abscess,  noma,  and  pseudotuberculosis. 

In  some  cases  the  actinomycetes  can  be  obtained  in  culture 
from  the  diseased  tissues;  almost  as  often  they  can  not. 
Sometimes  the  inoculation  of  animals  with  bits  of  the 
diseased  tissue  or  with  cultures  results  in  the  production 
of  pathological  lesions  referable  to  the  organism;  again 
no  effect  follows  upon  such  inoculation.  As  seen  in  the 
tissues  by  microscopic  examination,  actinomycetes  may 


ACTINOMYCETES  459 

appear  as  long,  convoluted,  irregularly  staining,  beaded, 
branching  threads,  or  as  clumps  of  short,  markedly  beaded, 
sometimes  branched  rods.  At  times  a  clump  of  the  short 
or  longer  threads  is  encountered  in  the  tissues  that  gives  the 
distinct  impression  of  mycelial  structure. 

Some  of  the  varieties  that  have  been  described  are  best 
demonstrated  in  the  tissues  or  exudates  by  the  Gram  or 
Gram-Weigert  method  of  staining;  others  are  decolorized 
by  this  process,  and  are  rendered  visible  only  by  the  simpler 
procedures.  Some  of  them  are  to  a  limited  extent  proof 
against  the  action  of  acid  decolorizers.  Though  many 
accounts  of  the  presence  of  these  morphological  types  in  a 
variety  of  conditions  have  been  recorded,  the  descriptions 
in  the  main  are  meagre  and  often  insufficient  for  identifi- 
cation. A  few,  however,  have  been  found  so  constantly  in 
association  with  more  or  less  definite  clinical  and  pathological 
conditions  that  a  brief  description  of  them  may  be  of  service. 

Actinomyces  Bovis  (also  commonly  known  as  streptothrix 
actinomyces,  actinomyces  fungus,  ray  fungus)  was  first 
observed  by  von  Langenbeck  in  a  case  of  vertebral  caries 
in  1845.  According  to  Bollinger,  the  fungus  had  been  seen 
by  Hahn  a  number  of  years  before  in  museum  specimens, 
but  had  been  regarded  by  him  as  a  penicillium.  The  name 
actinomyces  or  ray  fungus  originated  with  Harz. 

This  fungus  is  constantly  to  be  detected  in  the  tissues  and 
exuda'tes  of  the  disease  of  cattle  known  as  actinomycosis, 
"lumpy  jaw,"  "wooden  tongue,"  etc.  The  typical  tumor 
of  this  disease  is  characterized  by  inflammation,  pus  forma- 
tion, excessive  new  formation  of  connective  tissue,  abscesses, 
cavities  and  sinuses.  Viewed  as  a  whole,  the  tumor  pre- 
sents points  of  resemblance  to  the  osteosarcomatous,  to 
the  scrofulous  or  tuberculous,  and  to  the  cancerous  processes. 


460     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

The  disease  occasionally  occurs  in  man,  and  according  to  the 
point  of  entrance  of  the  parasite  may  arise  in  the  mouth,  the 
pharynx,  the  lungs,  the  intestines,  or  the  skin.  In  animals 
the  disease  is  characterized  by  an  excessive  new  forma- 
tion of  connective  tissue,  so  that  tumefaction  is  always  a 
conspicuous  peculiarity.  In  man,  on  the  other  hand, 
suppuration  is  the  most  prominent  feature. 

If  the  purulent  discharge  from  an  actinomycotic  tumor 
be  examined  fresh,  it  will  be  found  to  contain  tiny  yellow 

FIG.  79 


Actinomycosis  fungus  in  pus.     Fresh,  unstained  preparation.     Magnified 
about  500  diameters. 


(sulphur  color  as  a  rule)  clumps.  If  these  be  examined, 
unstained,  in  a  drop  of  physiological  salt  solution  or  water 
under  the  microscope,  they  will  be  found  to  be  made  up  of 
a  rosette-like  mass  of  closely  interwoven  threads.  (See 
Fig.  77.)  At  the  center  the  mass  may  show  the  presence  of 
spherical,  coccus-like  bodies  or  granules,  while  at  the  per- 
iphery the  free  ends  of  the  threads  are  more  or  less  distinctly 
bulbous  or  nodular,  or  both,  and  they  may  show  branching. 
Sometimes  the  free  ends  of  the  threads  are  only  slightly  or 
not  at  all  swollen. 


ACTINOMYCETES  461 

These  mycelia — the  actinomyces — may  be  stained  by 
the  ordinary  aniline  dyes,  or  by  the  Weigert  or  the  Gram 
method,  though  by  either  of  these  procedures  their  full  struct- 
ure is  not,  as  a  rule,  brought  out.  The  reason  for  this  is 
that  the  terminal  bulbs  are  not  due  to  enlargement  of  the 
thread  itself,  but  rather  to  a  colloid  degeneration  of  its 
enveloping  sheath.  This  colloid  matter,  having  different 
microchemical  reactions  from  the  enclosed  thread,  requires 
different  reagents  to  stain  it.  The  entire  structure  may  be 
seen  when  the  fungus  is  stained  first  by  the  Gram  method, 
and  subsequently  with  eo.sin  or  saffranin.  For  the  demon- 
stration of  the  fungus  in  sections,  the  method  of  Mallory 
gives  satisfaction.  It  is  as  follows;  Stain  the  section  on  the 
slide  with  gentian- violet;  clear  and  dehydrate  with  aniline 
oil  in  which  a  little  basic  fuchsin  has  been  dissolved;  remove 
the  aniline  oil-fuchsin  with  xylol,  and  mount  in  xylol  balsam. 
In  sections  treated  in  this  way  the  coccus-like  central 
masses  and  the  filamentous  threads  making  up  the  mass  of 
the  mycelium  are  stained  blue;  the  club-like  extremities  of 
the  thread  are  red.  Often  the  red-stained  hyaline  material 
is  seen  to  be  penetrated  to  its  extremity  by  a  sharply  defined 
blue  thread. 

Cultivation  of  the  fungus  from  the  actinomycotic  pus 
presents  difficulties  for  the  following  reasons:  Not  all  the 
mycelia  seen  by  microscopic  examination  are  living;  as  a 
rule  they  grow  slowly  even  under  the  best  of  circumstances; 
and  generally  there  are  many  other,  more  rapidly  growing, 
living  organisms  in  the  pus.  When  pure  cultures  are  ob- 
tained, it  grows  (according  to  Bostrom)  on  all  the  ordinary 
artificial  media.  It  develops  at  room-temperature,  but 
better  at  that  of  the  body. 

It  grows  both  with  and  without  oxygen. 


462     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

The  young  colonies  appear  as  grayish  points  composed  of 
a  felt-work  of  fine  threads.  As  the  colonies  become  older 
they  become  denser  and  more  opaque.  Very  old  colonies 
are  almost  leathery  in  consistency.  On  blood  serum  the 
growth  after  a  time  assumes  a  salmon,  an  orange,  or  a 
yellowish-red  color.  Growth  on  gelatin  is  accompanied  by 
slow  liquefaction. 

A  yellowish-red  growth,  limited  in  extent,  occurs  on 
potato.  It  causes  no  clouding  of  bouillon,  but  grows  as 
cottony  clumps  that  sink  to  the  bottom. 

The  bulbous  extremities  seen  upon  the  mycelial  threads 
fresh  from  the  pus  are  not  usually  seen  under  conditions 
of  artificial  cultivation.  They  are  sometimes  observed  in 
colonies  located  in  the  depths  of  solid  media.  The  white, 
powdery  coating  seen  on  old  colonies  represents  the  so-called 
"spores."  They  are  not,  however,  resistant  to  heat,  being 
destroyed,  according  to  Domec,  by  75°  C.  in  five  minutes. 

Bo  vines  are  the  animals  most  frequently  affected.  The 
disease  has  been  seen  in  swine,  dogs,  and  horses. 

The  most  common  seat  of  the  disease  is  the  jaw,  and  this, 
together  with  the  fact  that  particles  of  fodder,  such  as  bits 
of  grain,  chaff,  straw,  and  barley  beard,  have  been  detected 
in  the  diseased  tissues  in  association  with  the  causative 
fungus,  has  led  to  the  belief  that  the  parasite  gains  access 
to  the  tissues  with  such  foodstuffs.  It  has  not,  however, 
been  recognized  outside  the  animal  body.  The  disease  is 
apparently  not  transmissible  from  animal  to  animal  or  from 
animal  to  man.  Inoculation  of  animals  with  pure  cultures 
is  usually  negative,  although  nodular  formations  have  fol- 
lowed the  injection  of  large  quantities  into  the  peritoneal 
cavity  of  rabbits.  In  Bostrom's  cases  the  nodules  presented 
only  a  few  of  the  club-shaped  extremities  of  the  ^threads, 


ACTINOMYCETES  463 

and  there  was  no  evidence  of  multiplication  of  the  fungus; 
while  in  the  experiments  of  Israel  and  Wolf  it  is  said  there 
developed,  in  from  four  to  seven  weeks  after  intraperitoneal 
inoculation,  larger  and  smaller  tumors  in  which  typical 
mycelia  were  present,  and  from  which  the  fungus  was 
obtained  in  pure  culture. 

Actinomyces  Madurae. — This  organism  is  suppposed  to 
be  concerned  in  the  causation  of  mycetoma  or  Madura  foot. 
Two  varieties  of  mycetoma  are  known,  viz.,  the  pale  or 
ochroid  and  the  black  or  melanoid.  Save  for  its  occur- 
rence in  the  foot,  mycetoma  is  almost  a  counterpart  of 
actinomycosis;  and  the  suspicion  of  their  identity  is  by 
no  means  lessened  by  the  fact  that  the  actinomyces  con- 
stantly associated  with  the  ochroid  variety  is  to  all  intents 
and  purposes  identical  with  actinomyces  bo  vis.  It  differs 
from  that  organism  only  in  such  minor  details  as  to  leave 
little  doubt  that  they  are  very  closely  related,  if  not  iden- 
tical, so  that  a  description  of  the  one  serves  equally  to  aid 
in  the  identification  of  the  other. 

The  investigations  of  Wright,1  conducted  upon  a  case 
encountered  in  Boston,  point  to  another  type  of  parasite  as 
the  causative  factor  in  the  black  mycetoma.  Instead  of  an 
actinomyces,  Wright  found  a  true  mold.  He  expresses 
the  opinion  that  the  pale  mycetoma  is,  etiologically, 
actinomycosis,  and  that  the  black  is  a  hyphomycetic 
infection. 

The  fungus  encountered  and  isolated  in  pure  culture  by 
Wright  presented  the  following  characteristics:  As  ob- 
tained from  the  affected  tissues,  the  mycelia  under  the 
microscope  appear  as  black  or  brown  mulberry-like  masses 

1  A  Case  of  Mycetoma  (Madura  Foot),  Journal  of  Experimental  Medi- 
cine, 1898,  iii,  421. 


464     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

less  than  one  millimeter  in  diameter.  They  are  hard,  rather 
brittle,  and  difficult  to  break  up  under  the  cover-glass.  On 
soaking  them  in  a  strong  solution  of  sodium  hydroxide  they 
become  softened  and  the  structure  of  the  fungus-mass  can 
be  made  out.  Under  high  magnifying  power  these  masses 
are  found  to  consist  of  pigment  granules,  ovoid  translucent 
bodies,  and  distinctly  branching  separate  hyphse.  Some- 
times these  latter  exhibit  dilatations  or  varicosities  of  their 
segments.  The  periphery  of  a  fungous  mass  shows  the  pres- 
ence of  club-shaped  hyphse,  closely  set  and  radially  arranged. 
From  such  masses  growth  on  artificial  culture-media  may 
be  obtained.  When  transferred  direct  from  the  tissues  to 
artificial  media,  growth  in  every  case  starts  from  the  granule 
about  four  or  five  days  after  it  is  placed  upon  the  culture 
media. 

On  solid  media  it  first  appears  as  delicate  tufts  of  whitish 
filaments.  These  in  the  course  of  a  few  days  increase  in 
number  and  length,  and,  in  the  case  of  the  potato,  form  a 
dense  whitish  or  pale-brown  felt-work  having  a  tendency 
to  spread  widely. 

In  pure  cultivation  it  is  seen  as  long,  branching  hyphse 
with  delicate  transverse  septa.  In  old  forms  the  hyphse 
may  be  swollen  at  the  points  marked  by  the  septa,  and  may 
then  appear  as  strings  of  plump  oval  segments.  The  fila- 
ments have  a  definite  wall,  inclosing  granules  and  pale  areas. 
No  spore-bearing  organs  are  seen. 

On  potato,  it  grows  as  a  dense,  widely  spreading,  velvety 
membrane;  pale  brown  at  the  center  and  white  at  the 
periphery.  The  potato  takes  on  a  dark-brown  color  and 
becomes  very  moist  and  dark;  coffee-colored  granules 
appear  upon  the  surface  of  the  growth. 

In  bouillon  the  growth  assumes  a  puff-ball  appearance. 


ACTINOMYCETES  465 

The  medium  assumes  a  deep  coffee-brown  color,  and  ulti- 
mately a  mycelium  growth  appears  upon  the  surface  and 
throughout  the  fluid. 

When  grown  in  potato  infusion  (20  grams  of  potato 
boiled  in  water,  filtered  and  made  up  to  a  liter),  the  growth 
is  characterized  by  the  appearance  of  black  granules  in  the 
midst  of  the  mycelium.  The  black  granules  consist  of 
closely  packed  spherical  or  polyhedral  cells,  together  with 
some  short,  thick  segmented  hyphse.  The  walls  of  these 
cells  have  a  black  appearance,  and  masses  of  them  are  black 
and  opaque  under  the  microscope. 

On  agar-agar,  growth  appears  as  a  grayish  mesh-work  of 
widely  spreading  filaments.  In  old  cultures  black  granules 
(sclerotia)  appear  among  the  filaments.  No  growth  occurs, 
in  the  depth  of  the  medium. 

No  results  were  obtained  by  the  inoculation  of  animals 
with  either  the  material  direct  from  the  tissues  or  with  pure 
cultures. 

The  tissue  from  which  this  fungus  was  obtained  con- 
sisted, briefly,  of  a  more  or  less  atypical  connective- tissue 
new-growth,  with  numerous  areas  of  suppuration  marked  by 
the  presence  of  the  black  granules  just  described. 

On  histological  study  of  the  tumor  the  primary  effect* 
produced  by  the  parasite  appears  to  be  the  development 
of  nodules  of  epithelial  cells  and  of  giant  cells  from  the 
tissues  immediately  about  them.  Later,  suppuration  of  the 
nodules  and  abscess  formation  occur.  This  in  time  gives 
rise  to  excessive  development  of  granulation  and  connective 
tissue. 

Actinomyces    Farcinicus    (bacille    du    farcin    des    bceufs 
(Nocard);  oospora  farcinica;  actinomyces  bovis  farcinicus). 
— This  organism  was  discovered  by  Nocard   (1888)  in  a 
30 


466      APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

disease  of  cattle  that  is  suggestive  of  farcy  as  seen  in  horses. 
The  lesions  consist  of  chains  of  enlarged  subcutaneous 
lymph  glands,  which  on  examination  are  found  to  be  in  a 
condition  somewhat  simulating  tuberculosis.  Similar  nodules 
are  sometimes  encountered  in  the  internal  organs. 

By  microscopic  examination  the  organism  is  seen  as  long, 
branching  threads  consisting  of  short  segments. 

It  is  non-motile.  Spore-formation  is  questionable,  Nocard 
having  seen  it,  while  Lehmann  and  Neumann  have  not. 
The  organism  may  be  stained  by  the  ordinary  methods,  and 
also  by  the  Gram-Weigert  process.  It  grows  on  all  the 
ordinary  culture  media,  and  at  both  room-  and  body-tem- 
perature, especially  well  at  the  latter.  It  is  aerobic. 

Colonies  in  agar-agar  reach  a  size  of  from  1  to  2  mm.; 
are  yellowish-white  in  color,  irregular  in  outline,  and  have 
the  appearance  of  a  glazed,  membranous  mass. 

On  gelatin,  the  growth  is  much  slower,  so  that  after  ten 
days  the  colonies  appear  as  tiny  translucent  round  glistening 
points.  Under  low  power  of  the  microscope  these  colonies 
are  sharply  circumscribed,  grayish  or  greenish  in  color,  and 
are  without  characteristic  structure. 

Growth  in  bouillon  is  characterized  by  a  tough,  slimy 
sediment,  and  at  times  by  more  or  less  of  pellicle  formation. 
Pellicle  formation  is  encouraged  by  the  addition  of  glycerin. 
The  bouillon  is  not  uniformly  clouded  by  the  growth. 

In  milk,  it  causes  an  alkaline  reaction,  solution  of  casein, 
but  no  coagulation. 

On  potato,  it  grows  slowly  as  a  dull  yellowish-white  dry 
membrane. 

Bovines,  sheep,  and  guinea-pigs  are  susceptible  to  inocu- 
lation; rabbits,  dogs,  cats,  horses,  and  asses  are  not. 


ACTINOMYCETES  467 

When  pure  cultures  are  injected  into  either  the  circulation 
or  the  peritoneal  cavity  of  guinea-pigs,  death  ensues  in 
from  nine  to  twenty  days.  The  autopsy  reveals  diffuse 
pseudotuberculosis  of  the  omentum.  Within  the  pseudo- 
tubercles  the  organism  is  seen  as  long,  branching  threads, 
often  matted  together  as  a  true  mycelium. 

By  subcutaneous  inoculation  only  the  neighboring  lymph- 
glands  are  affected. 

The  disease  farcin  des  bceufs  is  said  to  be  more  common 
in  Guadeloupe  than  elsewhere. 

Actinomyces  Eppingeri. — This  organism  was  discovered  by 
Eppinger  in  an  abscess  of  the  brain.  He  regarded  it  as  a 
cladothrix,  and  gave  to  it  the  designation  cladothrix  aster- 
oides.  It  grows  well  in  pure  culture  under  artificial  con- 
ditions, and  is  pathogenic  for  animals.  In  the  case  studied 
by  Eppinger  the  organism  was  present  not  only  in  the 
abscess,  but  also  in  the  meninges  of  the  brain  and  cord  and 
in  the  bronchial  and  supraclavicular  lymph  glands.  There 
is  no  doubt  of  its  causal  relation  to  the  conditions. 

In  pure  culture  it  grows  well  on  ordinary  media.  It 
appears  as  long,  branching  threads,  many  of  which  are  com- 
posed of  short  quadratic  segments.  Spores  are  not  formed. 
Motility  is  doubtful;  it  has  been  observed  by  Eppinger, 
while  Lehmann  and  Neumann  failed  to  detect  it.  It  stains 
both  by  the  ordinary  dyes  and  by  the  method  of  Gram.  It 
grows  scarcely,  if  at  all,  under  anaerobic  conditions.  It 
grows  at  room-temperature,  but  much  better  at  the  tem- 
perature of  the  body.  The  best  growth  is  observed  on 
nutrient  agar-agar  containing  2  per  cent,  of  glucose.  The 
colonies  on  the  surface  of  glucose-agar-agar  appear  as 
yellowish-white,  round,  finely  granular,  dull  patches  that  are 


468     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

surrounded  by  a  narrow  paler  zone.  In  the  depths  of  the 
medium  they  do  not  develop  beyond  very  small  points. 

On  gelatin  the  growth  is  very  slow;  there  is  no  lique- 
faction, and  after  a  time  the  colonies  take  on  an  orange-red 
color. 

Bouillon  is  not  uniformly  clouded.  Growth  takes  place 
on  the  surface  in  the  form  of  a  whitish  pellicle,  in  which 
dense  white  masses  may  be  seen.  These  latter  increase  in 
size,  become  detached,  and  fall  to  the  bottom  of  the  vessel, 
to  collect  as  mycelium-like  sediment. 

On  potato,  growth  begins  as  a  coarsely  granulated  white 
layer,  which  becomes  gradually  red  in  color.  It  is  ultimately 
covered  by  a  fine,  hair-like  growth. 

Both  rabbits  and  guinea-pigs  are  susceptible  to  its  patho- 
genic action.  When  injected  into  either  the  circulation,  the 
peritoneal  cavity,  or  beneath  the  skin,  there  develop  in  from 
one  to  four  weeks  a  condition  closely  simulating  tubercu- 
losis ("  pseudotuberculosis  cladothrica")-  The  organism 
quickly  loses  its  pathogenic  properties  under  artificial 
cultivation. 

Actinomyces  Pseudotuberculosis. — In  1897  Flexner  detected 
this  organism  in  a  consolidated  and  caseous  lung.  The  con- 
dition suggested  tuberculosis.  The  lesion  consisted  mainly 
of  an  inflammatory  exudation  that  had  undergone  casea- 
tion,  but  in  addition  there  were  present  isolated  nodules 
that  in  size  and  general  appearance  were  difficult  to  distin- 
guish from  miliary  tubercles.  Giant  cells  were  not  seen. 
The  streptothrix  was  abundant  in  the  lung,  appearing  as 
masses  of  convoluted,  branching  threads.  The  contours  of 
the  rods  were  not  quite  uniform,  the  staining  was  irregular, 
and  occasionally  a  thread  was  seen  that,  toward  its  extrem- 


ACTINOMYCETES  469 

ity,  appeared  to  be  breaking  up  into  short  segments.  No 
coccus-like  forms  were  '  seen.  It  is  stained  best  by  the 
Weigert  method,  when  deeply  stained  masses  separated 
from  one  -another  by  more  or  less  clear  spaces  are  to  be 
detected.  The  organism  was  not  obtained  in  culture,  and  no 
effect  was  produced  on  guinea-pigs  by  subcutaneous  inocu- 
lation with  bits  of  the  diseased  lung. 


CHAPTER  XXII. 

Glanders — Characteristics  of  the  Disease — Histological  Structure  of  the 
Glanders  Nodule — Susceptibility  of  Different  Animals  to  Glanders — 
The  Bacterium  of  Glanders;  Its  Morphological  and  Cultural  Pecu- 
liarities— Diagnosis  of  Glanders — Mallein. 

THE  disease  is  generally  known  as  glanders  when  the 
mucous  membrane  of  the  nostrils  is  affected,  and  as  farcy 
when  the  subcutaneous  lymphatics  are  the  principal  sites 
of  involvement. 

Though  most  commonly  seen  in  the  horse  and  ass, 
glanders  is  not  rarely  met  with  in  other  animals,  and 
is  occasionally  encountered  in  man.  When  occurring 
in  the  horse  its  primary  seat  is  usually  upon  the  mucous 
membrane  of  the  nostrils.  It  appears  in  the  form  of 
small  gray  nodules,  about  which  the  membrane  is  con- 
gested and  swollen.  These  nodules  ultimately  coalesce  to 
form  ulcers.  There  is  a  profuse  slimy  discharge  from  the 
nostrils  during  the  course  of  the  disease.  The  primary 
lesion  may  extend  from  its  seat  in  the  nose  to  the  mouth, 
larynx,  trachea,  and  ultimately  to  the  lungs.  Its  secondary 
manifestations  are  observed  along  the  lymphatics  that  com- 
municate with  the  initial  focus;  in  the  lymphatic  glands,  and 
as  metastatic  foci  in  the  internal  organs. 

Less  frequently  the  disease  is  seen  to  begin  beneath  the 
skin,  particularly  in  the  region  of  the  neck  and  breast.  When 
in  this  locality  the  subcutaneous  lymphatics  become  in- 
volved, and  are  converted  into  indurated,  knotty  cords — 
"farcy-buds" — easily  discernible  from  without. 
(470) 


GLANDERS  471 

In  man  it  usually  occurs  in  individuals  who  have  been 
in  attendance  upon  animals  affected  with  the  disease.  It 
may  occur  upon  the  mucous  membrane  of  the  nares;  but 
its  most  frequent  expressions  are  in  the  skin  and  muscles, 
where  appear  abscesses,  phlegmons,  erysipelas-like  inflam- 
mations, and  local  necrosis  closely  resembling  carbuncles. 
Metastases  to  the  lungs,  kidneys,  and  testicles,  as  in  the 
horse,  may  also  be  seen. 

When  occurring  upon  the  mucous  membrane  glanders  is 
characterized  by  the  presence  of  gray  nodules,  about  as 
large  as  a  pin-head,  that  closely  resemble  miliary  tubercles 
in  their  naked-eye  appearance.  These  consist  histologically 
of  granulation-tissue — i.  e.,  of  small  round  cells,  very  similar 
to  proliferating  leukocytes — of  some  lymph  cells,  and,  in  the 
earliest  stages,  of  a  small  portion  of  necrotic  tissue.  As 
they  grow  older,  and  the  process  advances,  there  is  a  tendency 
to  central  necrosis,  with  the  ultimate  formation  of  a  soft, 
yellow,  creamy,  pus-like  material.  Though  strikingly  like 
miliary  tubercles  in  certain  respects  in  the  early  stages, 
they  present,  nevertheless,  decided  points  of  difference  when 
examined  more  in  detail. 

The  round-cell  infiltration  of  the  glanders  nodule  consists 
essentially  of  polymorphonuclear  leukocytes,  while  that  of 
the  miliary  tubercle  partakes  more  of  the  nature  of  a  lym- 
phocytic  infiltration;  in  the  later  stages  of  the  process  the 
glanders  nodule  breaks  down  into  a  soft,  creamy  matter, 
very  analogous  to  ordinary  pus,  while  in  the  later  stages 
of  the  miliary  tubercle  the  tendency  is  to  an  'amalgamation 
of  its  histological  constituents,  and  ultimately  to  necrosis 
with  caseation.  The  giant-cell  formation  common  to  tuber- 
culosis is  never  seen  in  the  glanders  nodule.  As  Baumgarten 
aptly  puts  it:  "The  pathological  manifestations  of  glanders, 


472      APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

from  the  histological  aspect,  stand  midway  between  the 
acute  purulent  and  the  chronic  inflammatory  processes."1 
Evidently  these  differences  are  only  to  be  explained  by  dif- 
ferences in  the  nature  of  the  causes  that  underlie  the  several 
affections.  We  have  studied  the  characteristics  of  bacterium 
tuberculosis;  we  shall  now  take  up  the  bacillus  of  glanders 
and  note  the  striking  differences  between  them. 

BACTERIUM  MALLEI  (LOFFLER),  MIGULA,  1900. 

SYNONYMS:  Bacillus  mallei  (Loffler),  1886;  Rotz  bacillus,  Kranzfeld, 
1887. 

In  1882  Loffler  and  Schiitz  discovered  in  the  diseased 
tissues  of  animals  suffering  from  glanders  a  bacterium  that, 
when  isolated  in  pure  culture  and  inoculated  into  susceptible 
animals,  possesses  the  property  of  reproducing  the  disease 
with  all  its  clinical  and  pathological  manifestations.  It  is 
therefore  the  cause  of  the  disease. 

This  organism  is  a  rod,  with  rounded  or  slightly  pointed 
ends.  It  usually  stains  somewhat  irregularly.  (See  Fig. 
78.)  When  examined  in  stained  preparations  its  continuity 
is  marked  by  alternating  darkly  and  lightly  stained  areas. 
It  is  usually  seen  as  a  single  rod,  but  may  occur  in  pairs, 
and  less  frequently  in  longer  filaments.  ,. 

The  question  as  to  its  spore-forming  property  is  still  an 
open  one,  though  the  weight  of  evidence  is  in  opposition 
to  the  opinion  that  it  possesses  this  peculiarity.  Certain 
observers  claim  to  have  demonstrated  spores  in  the  bacteria 
by  particular  methods  of  staining;  but  this  statement  can 

1  For  a  further  discussion  of  the  pathology  and  pathogenesis  of  this 
disease,  see  Lehrbuch  der  pathologischen  Mykologie,  by  Baumgarten, 
1890.  See,  also,  Wright,  The  Histological  Lesions  of  Acute  Glanders  in 
Man,  Journal  of  Experimental  Medicine,  i,  577. 


BACTERIUM  MALLEI 


473 


have  but  little  weight  when  compared  with  the  behavior 
of  the  organism  when  subjected  to  more  conclusive  tests. 
For  example,  it  does  not,  at  any  stage  of  development,  resist 
exposure  to  3  per  cent,  carbolic  acid  solution  for  longer  than 
five  minutes,  nor  to  1 : 5000  sublimate  solution  for  more  than 
two  minutes.  It  is  destroyed  in  ten  minutes  in  some  experi- 
ments, and  in  five  in  others,  by  a  temperature  of  55°  C.; 


FIG.  80 


Bacterium  mallei,  from  culture. 

and  when  dried  it  loses  its  vitality,  according  to  different 
observers,  in  from  thirty  to  forty  days;  all  of  which  speak 
directly  against  this  being  a  spore-bearing  bacillus. 

It  is  not  motile,  and  does  not  therefore  possess  flagella. 

It  grows  readily  on  ordinary  nutrient  media  at  from  25° 
to  38°  C. 

Upon  nutrient  agar-agar,  both  with  and  without  glycerin, 
it  appears  as  a  moist,  opaque,  glazed  layer,  with  nothing 


474     APPLICATION  OF  METHODS  OF   BACTERIOLOGY, 

characteristic  about  it.  This  is  true  both  for  smear  cultures 
and  for  single  colonies. 

Its  growth  on  gelatin  is  much  less  voluminous  than  on 
media  that  can  be  kept  at  higher  temperature,  though  it 
does  grow  on  this  medium  at  room-temperature  without 
causing  liquefaction. 

Its  growth  on  blood  serum  is  in  the  form  of  a  moist, 
opaque,  slimy  layer,  inclining  to  a  yellowish  or  dirty, 
brownish-yellow  tinge.  It  does  not  liquefy  the  serum. 

On  potato  its  growth  is  moderately  rapid,  appearing  in 
from  twenty-four  to  thirty-six  hours  at  37°  C.  as  a  moist, 
amber-yellow,  transparent  deposit  having  somewhat  the 
appearance  of  honey;  this  becomes  deeper  in  color  and 
denser  in  consistence  as  growth  progresses,  and  finally  takes 
on  a  reddish-brown  color;  at  the  same  time  the  potato 
about  it  becomes  darkened. 

In  bouillon  it  causes  diffuse  clouding,  with  ultimately  the 
formation  of  a  more  or  less  tenacious  or  ropy  sediment. 

In  milk  to  which  a  little  litmus  has  been  added  it  causes 
the  blue  color  to  become  red  or  reddish  in  from  four  to  five 
days,  and  quite  red  after  two  weeks  at  37°  C.  At  the  same 
time  the  milk  separates  into  clear  whey  and  a  firm  clot  of 
casein. 

Its  reactions  to  heat  are  very  interesting.  At  42°  C.  it 
will  often  grow  for  twenty  days  or  more.  It  will  not  grow 
at  43°  C.,  and  if  exposed  to  this  temperature  for  forty-eight 
hours  it  is  destroyed.  It  is  killed  in  five  hours  when  exposed 
to  50°  C.,  and  in  five  minutes  by  55°  C. 

It  grows  both  with  and  without  oxygen;  it  is  therefore 
facultative  as  regards  its  relation  to  this  gas. 

On  cover-slips  it  stains  readily  with  all  the  basic  aniline 
dyes,  and,  as  a  rule,  as  stated,  presents  conspicuous  irregu- 


BACTERIUM  MALLEI  475 

larities  in  the  way  that  it  takes  up  the  dyes,  being  usually 
marked  by  deeply  stained  areas  that  alternate  with  points 
at  which  it  either  does  not  stain  at  all  or  only  slightly. 

The  animals  susceptible  to  infection  by  this  organism  are 
horses,  asses,  field-mice,  guinea-pigs,  and  cats.  Baumgarten 
records  cases  of  infection  in  lions  and  tigers  that  were  fed, 
in  menageries,  with  flesh  from  horses  affected  with  the 
disease.  Rabbits  are  but  slightly  susceptible;  dogs  and 
sheep  still  less  so.  Man  is  susceptible,  and  infection  not 
rarely  terminates  fatally.  White  mice,  common  gray  house- 
mice,  rats,  cattle,  and  hogs  are  insusceptible. 

Inoculation  Experiments. — The  most  favorable  animal 
upon  which  to  study  the  pathogenic  properties  of  this 
organism  in  the  laboratory  is  the  common  field-mouse. 
When  inoculated  subcutaneously  with  a  small  portion  of  a 
pure  culture  of  bacterium  mallei  death  ensues  in  about 
seventy-two  hours.  The  most  conspicuous  tissue  changes 
will  be  enlargement  of  the  spleen,  which  is  at  the  same  time, 
almost  constantly,  studded  with  minute  gray  nodules,  the 
typical  glanders  nodule.  They  are  rarely  present  in  the 
lungs,  but  may  frequently  be  seen  in  the  liver.  From  these 
nodules  the  glanders  bacillus  may  be  obtained  in  pure  culture. 
With  the  exception  of  the  characteristic  nodules,  the  disease 
as  seen  in  this  animal  presents  none  of  the  features  that 
it  displays  in  the  horse  and  ass.  The  clinical  and  patho- 
logical manifestations  resulting  from  inoculation  of  guinea- 
pigs  are  much  more  faithful  reproductions.  The  animal 
lives  usually  from  six  to  eight  weeks  after  inoculation,  and 
during  this  time  becomes  affected  with  a  group  of  most 
interesting  and  peculiar  pathological  processes.  The  specific 
inflammatory  condition  of  the  mucous  membrane  of  the 
nostrils  is  almost  always  present.  The  joints  become  swollen 


476     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

and  infiltrated  to  such  an  extent  as  often  to  interfere  with 
the  use  of  the  legs.  In  male  animals  the  testicles  become 
enormously  distended  with  pus,  and  on  closer  examination 
a  true  orchitis  and  epididymitis  are  seen  to  be  present. 
The  internal  organs,  particularly  the  lungs,  kidneys,  spleen, 
and  liver,  are  usually  the  seat  of  the  nodular  formations 
characteristic  of  the  disease.  From  all  of  these  disease-foci 
the  bacillus  causing  them  ca.n  be  isolated  in  pure  culture. 

Staining  in  Tissues. — Though  always  present  in  the  diseased 
tissues,  considerable  trouble  is  usually  experienced  in  demon- 
strating the  bacteria  by  staining  methods.  The  difficulty  is 
due  to  the  fact  that  the  bacteria  are  very  easily  decolorized, 
and  in  tissues  stained  by  the  ordinary  processes  are  robbed 
of  their  color  even  by  the  alcohol  with  which  the  tissue  is 
rinsed  and  dehydrated.  If  we  will  remember  not  to  employ 
concentrated  stains,  and  not  to  expose  the  sections  to  the 
stains  for  too  long  a  time,  but  little  treatment  with  decolor- 
izing agents  is  necessary,  and  very  satisfactory  preparations 
will  be  obtained.  A  number  of  methods  have  been  suggested 
for  staining  the  glanders  bacilli  in  tissues,  and  if  what  has 
been  said  will  be  borne  in  mind,  no  difficulty  should  be 
experienced.  Two  satisfactory  methods  that  we  have  used 
for  this  purpose,  though  perhaps  no  better  than  some  of  the 
others,  are  as  follows : 

a.  Transfer  the  sections  from  alcohol  to  distilled  water. 
This  lessens  the  intensity  with  which  the  stain  subsequently 
takes  hold  of  the  tissues,  by  diminishing  the  activity  of  the 
diffusion  that  would  occur  if  they  were  placed  froin  alcohol 
into  watery  solutions  of  the  dyes.  Transfer  from  distilled 
water  to  the  slide,  absorb  all  water  with  blotting-paper, 
and  stain  with  two  or  three  drops  of 

Carbol-fuchsin 10  c.c. 

Distilled  water    .  ...      100  c.c. 


BACTERIUM  MALLEI  477 

for  thirty  minutes;  absorb  all  superfluous  stain  with  blot- 
ting-paper, and  wash  the  section  three  times  with  0.3  per 
cent,  acetic  acid,  not  allowing  the  acid  to  act  for  more  than 
ten  seconds  each  time.  Remove  all  acid  from  the  section 
by  carefully  washing  in  distilled  water;  absorb  all  water 
by  gentle  pressure  with  blotting-paper;  and  finally,  at  wry 
moderate  heat,  or  with  a  small  bellows  (Kiihne),  dry  the  section 
completely  on  the  slide.  When  dried  clear  in  xylol  and  mount 
in  xylol  balsam. 

6.  Transfer  the  sections  from  alcohol  to  distilled  water; 
from  water  to  the  dilute  fuchsin  solution,  and  gently  warm 
(about  50°  C.)  for  fifteen  to  twenty  minutes.  Transfer 
sections  from  the  staining-solution  to  the  slide,  absorb  all 
superfluous  stain  with  blotting-paper,  and  then  treat  them 
with  1  per  cent,  acetic  acid  from  one-half  to  three-quarters 
of  a  minute.  Remove  all  trace  of  acid  with  distilled  water, 
absorb  all  water  by  gentle  pressure  with  blotting-paper,  and 
then  treat  the  sections  with  absolute  alcohol  by  allowing 
it  to  flow  over  them  drop  by  drop.  For  small  sections  three 
or  four  drops  are  sufficient.  Under  no  circumstances  should 
the  alcohol  be  allowed  to  act  for  more  than  one-quarter  of 
a  minute.  Clear  in  xylol  and  mount  in  xylol  balsam. 

By  method  b  the  tissues  are  better  preserved  than  by 
method  a,  by  which  they  are  dried. 

In  properly  stained  tissues  the  bacteria  will  be  found 
most  numerous  in  the  center  of  the  nodules,  becoming  fewer 
as  we  approach  the  periphery.  They  usually  lie  between 
the  cells,  but  at  times  may  be  seen  almost  filling  some  of 
the  epithelial  cells,  of  which  the  nodule  contains  more  or  less. 
They  are  always  present  in  these  nodules  in  the  tissues;  they 
are  rarely  present  in  the  blood,  and,  if  so,  in  only  small 
numbers. 


478     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

Diagnosis  of  the  Disease  by  Agglutination  and  Complement- 
fixation.  The  quickest  and  surest  method  of  recognizing  the 
disease  is  by  the  specific  agglutinating  effect  of  the  serum  of 
the  diseased  animal  upon  the  organism  of  the  disease.  Many 
different  plans  have  been  recommended.  That  of  Moore,  of 
Cornell  University,  is  one  of  the  most  trustworthy.  He 
recommends  a  test  emulsion  made  by  suspending  a  glycerin- 
agar  culture  of  glanders  bacilli  in  physiological  salt  solution. 
This  is  then  exposed  to  60°  C.  for  two  hours,  whereby  the 
bacteria  are  killed,  and  is  finally  preserved  by  the  addition 
of  0.5  per  cent,  carbolic  acid.  To  this  suspension  the  serum 
of  the  suspected  animal  is  added  in  varying  proportions 
until  a  distinct  clumping  and  sedimentation  of  the  bacteria 
is  observed.  Whenever  done  in  a  small  test-tube  of  about 
0.5  cm.  diameter  this  reaction  manifests  itself  as  a  gradual 
clarification  of  the  milky  fluid  and  the  accumulation  of  a  mass 
on  the  bottom  of  the  tube.  Normal  horse  serum  in  a  dilution 
of  1  to  300  to  1  to  200  causes  the  agglutination,  while  that 
from  glanders  animals  does  the  same  in  from  1  to  3200  to 
1  to  500  dilution. 

The  "complement-fixation"  reaction  may  also  be  applied 
both  for  the  recognition  of  the  condition — i.  e.,  for  detecting 
the  specific  antibodies  in  the  tissues  or  fluids,  as  well  as  for 
the  identification  of  the  specific  exciter  of  the  condition — 
i.  e.,  the  antigen.  (See  that  reaction.) 

Mallein. — The  sterile  filtered  products  of  growth  of  the 
glanders  bacillus  in  fluid  media  represent  what  is  known  as 
mallein — a  solution  of  compounds  that  bear  to  glanders  a 
relation  analogous  to  that  which  tuberculin  bears  to  tuber- 
culosis. It  is  used  with  considerable  success  as  a  diagnostic 
aid  in  detecting  the  existence  or  absence  of  deep-seated 
manifestations  of  the  disease,  the  glanderous  animal  reacting 


BACTERIUM  MALLEI  479 

(manifested  by  elevations  of  body-temperature  greater  than 
1.5°  C.)  to  subcutaneous  injections  of  mallein  in  from  four 
to  ten  hours,  while  an  animal  not  so  affected  gives  no  such 
reactions. 

Mallein  is  prepared  from  old  glycerin-bouillon  cultures  of 
the  glanders  bacterium  by  steaming  them  for  several  hours  in 
the  sterilizer,  after  which  they  are  filtered  through  unglazed 
porcelain. 

By  some  it  is  said  that  the  repeated  injection  of  mallein 
in  small  doses  confers  immunity  from  infection  by  bacterium 
mallei  upon  animals  so  treated;  an  opinion  that  is  entirely 
in  accord  with  the  principles  underlying  the  artificial  induc- 
tion of  immunity  in  general. 


CHAPTER  XXIII. 

Bacterium  (Syn.  Bacillus)  Diphtherias — Its  Isolation  and  Cultivation — 
Morphological  and  Cultural  Peculiarities — Pathogenic  Properties — 
Variations  in  Virulence — Bacterium  Pseudodiphtheriticum — Bacterium 
Xerosis — Diphtheria  Antitoxin. 

FROM  the  gray-white  deposit  on  the  fauces  of  a  diph- 
theritic patient  prepare  a  series  of  cultures  in  the  following 
way: 

Have  at  hand  five  or  six  tubes  of  Loffler's  blood-serum 
mixture.  (See  chapter  on  Media.)  Pass  a  stout  platinum 
needle,  which  has  been  sterilized,  into  the  membrane  and 
twist  it  around  once  or  twice,  or  brush  it  gently  over  the 
surface  of  the  membrane.  Without  touching  it  against 
anything  else  rub  it  carefully  over  the  surface  of  one  of  the 
serum  tubes;  without  sterilizing  it  pass  it  over  the  surface 
of  the  second,  then  the  third,  fourth,  and  fifth  tubes.  Place 
these  tubes  in  the  incubator.  Then  prepare  cover-slips 
from  scrapings  from  the  membrane  on  the  fauces.  If  the 
case  is  one  of  true  diphtheria,  the  tubes  will  be  ready  for 
examination  on  the  following  day. 

The  reason  that  plates  are  not  made  in  the  regular  way 
in  this  examination  is  that  the  bacillus  of  diphtheria  develops 
much  more  luxuriantly  on  the  serum  mixture,  from  which 
plates  cannot  be  made,  than  it  does  on  the  media  from  which 
they  can  be  made.  The  method  employed,  however,  insures 
a  dilution  in  the  number  of  organisms  present,  and  this,  in 
addition  to  the  fact  that  the  blood  serum  mixture  is  a  much 
more  favorable  medium  for  the  rapid  development  of  the 
diphtheria  organism  than  of  the  other  organisms  present, 
(480) 


BACTERIUM  DIPHTHERIA  481 

makes  its  isolation  by  this  method  a  matter  of  but  little 
difficulty. 

After  twenty-four  hours  in  the  incubator  the  tubes  present 
a  characteristic  appearance.  Their  surfaces  are  marked  by 
more  or  less  irregular  patches  of  a  white  or  cream-colored 
growth,  which  is  usually  more  dense  at  the  center  than  at 
the  periphery.  Except  now  and  then,  when  a  few  orange- 
colored  colonie^  may  be  seen,  these,  large  irregular  patches 
are  the  conspicuous  objects  on  the  surface  of  the  serum. 
Occasionally,  almost  nothing  else  appears. 

The  cover-slips  made  from  the  membrane  at  the  time  the 
cultures  were  prepared  will  be  found  on  microscopic  examina- 
tion to  present,  in  many  cases,  a  great  variety  of  organisms; 
but  conspicuous  among  them  will  be  noticed  slightly  curved 
bacilli  of  irregular  size  and  outline.  In  some  cases  they  will 
be  more  or  less  clubbed  at  one  or  both  ends;  sometimes 
they  appear  spindle  in  shape,  again  as  curved  wedges;  now 
and  then  they  are  irregularly  segmented.  They  are  rarely 
or  never  regular  in  outline.  If  the  preparation  has  been 
stained  with  Loffler's  alkaline  methylene-blue  solution,  many 
of  these  irregular  rods  are  seen  to  be  marked  by  circumscribed 
points  in  their  protoplasm  which  stain  very  intensely — 
they  appear  almost  black.  This  irregularity  in  outline  is 
the  morphological  characteristic  of  bacillus  diphtherise  of 
Loffler,  the  most  pleomorphic  organism  with  which  we  have 
to  deal. 

It  must  be  remembered,  however,  that  the  diagnosis 
of  diphtheria  should  not  under  all  circumstances  be  made 
from  the  examination  of  cover-slip  preparations  alone,  espe- 
cially when  they  are  stained  only  by  the  usual  method — 
i.  e.,  with  Loffler's  methylene-blue.  There  are  other  organ- 
isms present  in  the  mouth  cavity,  particularly  in  the  mouths 
31 


482      APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

of  persons  having  decayed  teeth,  the  morphology  of  which 
is  so  like  that  of  the  bacillus  of  diphtheria  that  they  might 
easily  be  mistaken  for  that  organism  if  subjected  to  only 
the  usual  method  of  microscopic  examination;  and  again, 
the  genuine  diphtheria  organism  is  sometimes  found  in  the 
mouth  cavities  of  healthy  persons  in  attendance  upon  diph- 
theria cases,  such  persons  being  at  the  time  insusceptible 
to  the  pathogenic  activities  of  the  organism^  In  the  vast 
majority  of  instances,  however,  where  the  clinical  condition 
of  the  patient  justifies  a  suspicion  of  diphtheria,  a  micro- 
scopic examination  alone  of  the  deposit  in  the  throat,  made 
by  an  experienced  person,  will  serve  to  confirm  or  contradict 
this  opinion,  and  such  examinations  very  frequently  reveal 
the  diphtheritic  nature,  etiologically  speaking,  of  mild  con- 
ditions of  the  throat  which  are  not  associated  with  grave 
constitutional  manifestations. 

BACTERIUM  DIPHTHERIA  (LOFFLER),  MIGULA,  1900. 

SYNONYMS:  Bacillus  diphtherias,  Loffier,  1884;  Klebs-Loffler  bacillus; 
Corynebacterium  diphtheriae,  Lehmann  and  Neumann,  1896. 

Bacterium  diphtherise,  discovered  microscopically  by 
Klebs,  and  isolated  in  pure  culture  and  proved  to  stand  in 
causal  relation  to  diphtheria  by  Loftier,  can  readily  be 
identified  by  its  cultural  peculiarities  and  by  its  pathogenic 
activity  when  introduced  into  tissues  of  susceptible  animals. 
In  guinea-pigs  and  kittens  the  results  of  its  growth  are  his- 
tologically  identical  with  those  found  in  the  bodies  of  human 
beings  who  have  died  of  diphtheria. 

When  studied  in  pure  culture  its  morphological  and  cul- 
tural peculiarities  are  as  follows : 

Morphology. — As  obtained  directly  from  the  diphtheritic 
deposit  in  the  throat  of  an  individual  sick  of  the  disease, 


BACTERIUM  DIPHTHERIAS  483 

it  is  sometimes  comparatively  regular  in  shape,  appearing 
as  straight  or  slightly  curved  rods  with  more  or  less  pointed 
ends.  More  frequently,  however,  spindle-  and  club-shapes 
occur,  and  not  rarely  many  of  these  rods  stain  irregularly; 
in  some  of  them  very  deeply  stained  round  or  oval  points 
can  be  detected. 

When  cultures  are  examined  microscopically  it  is  especially 
characteristic  to  find  irregular,  bizarre  forms,  such  as  rods 
with  one  or  both  ends  swollen,  and  very  frequently  rods 
broken  at  irregular  intervals  into  short,  sharply  defined 
segments,  either  round,  oval,  or  with  straight  sides.  Some 
forms  stain  uniformly,  others  in  various  irregular  ways,  the 
most  common  being  the  appearance  of  deeply  stained 
granules  in  a  lightly  stained  bacillus. 

By  a  series  of  studies  upon  this  organism  when  cultivated 
under  artificial  conditions  it  has  been  found  that  its  form  and 
size  depend  very  largely  upon  the  nature  of  its  environment. 
That  is  to  say,  its  morphology  is  always  more  regular,  and 
it  is  smaller  on  glycerin-agar-agar  than  on  other  media  used 
for  its  cultivation;  while  upon  Loffler's  blood  serum  the 
other  extremes  of  development  appear:  here  one  sees, 
instead  of  the  very  short,  spindle-,  lancet-,  club-shaped, 
always  segmented  and  regularly  staining  forms  as  seen  upon 
glycerin-agar-agar,  long,  sometimes  extremely  slender,  some- 
times thicker,  irregularly  staining  threads  that  may  be  either 
clubbed  or  pointed  at  their  extremities.  They  are,  as  a  rule, 
marked  by  areas  that  stain  more  intensely  than  does  the 
rest  of  the  rod,  and  at  times  they  may  be  a  little  swollen 
at  the  center.  These  differences  are  so  conspicuous  that 
microscopic  preparations  from  cultures  from  the  same  source, 
but  cultivated  in  the  one  case  on  glycerin-agar-agar  and  in 
the  other  upon  blood  serum,  when  placed  side  by  side  would 


484     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

hardly  be  recognized  as  of  the  same  organism,  unless  its 
peculiar  behavior  under  these  circumstances  was  already 
known.  Another  peculiar  variation  is  that  observed  upon 
very  slightly  acid  blood  serum.  Here  the  rods  appear 
swollen,  and  are  usually  contracted  to  oval  or  short,  oblong 
bodies,  which  stain  very  faintly,  and  in  which  are  usually 
located  one  or  two  very  deeply  staining  round  or  oval  points. 
Various  authors  have  called  attention  to  branching  forms 
of  this  organism  that  are  occasionally  encountered,  especially 
when  cultivated  upon  albumin.  We  have  never  seen  the 
branching  diphtheria  organisms  under  conditions  that  might 
reasonably  be  regarded  as  favorable  to  normal  development; 
and  in  many  thousand  blood  serum  cultures  from  cases  of 
diphtheria  that  have  been  examined  by  competent  bacteriol- 
ogists at  the  laboratory  of  the  Bureau  of  Health  of  Philadel- 
phia, the  branching  forms  of  this  organism  have  not  been 
observed  in  a  single  instance.  It  is  fair  to  assume,  there- 
fore, that  this  peculiar  morphological  variation  of  bacillus 
diphtheriae  is,  under  normal  conditions  of  growth,  com- 
paratively rare. 

On  the  other  hand,  if  the  organism  be  grown  on  media 
favorable  to  involution,  such,  for  instance,  as  hard-boiled  egg, 
or  coagulated  egg  of  slightly  acid  reaction,  branching  may  be 
seen,  but  with  it  degenerated  organisms  are  so  conspicuous 
as  to  leave  no  doubt  that  the  so-called  branching  and  involu- 
tion are  attributable  to  the  same  cause,  namely,  unsuitable 
conditions  of  cultivation. 

On  plain  nutrient  agar-agar  (that  is,  nutrient  agar-agar 
without  glycerin);  on  a  medium  consisting  of  dried  com- 
mercial albumin  dissolved  in  bouillon  (about  10  grams 
of  albumin  to  100  c.c.  of  bouillon  containing  1  per  cent,  of 
grape-sugar);  in  bouillon  without  glycerin,  and  in  bouillon 


BACTERIUM  DIPHTHERIA 


485 


to  which  a  bit  of  hard-boiled  egg  has  been  added,  the  mor- 
phology of  the  organism  is  about  intermediate,  in  both 
size  and  outlines,  between  the  forms  seen  upon  glycerin- 
agar-agar  and  upon  Loffler's  blood-serum.  There  will 
appear  about  an  equal  number  of  short  segmented  and 
longer,  irregularly  staining  forms;  but  in  general  the  longest 

FIG.  81 


A. 


Bacterium  diphtheria.  A,  its  morphology  on  glycerin-agar-agar;  B, 
its  morphology  on  Loffler's  blood-serum;  (7,  its  morphology  on  acid  blood- 
serum  mixture. 


are  rarely  as  long  as  the  long  forms  seen  on  blood-serum, 
and  throughout  they  are  not  so  conspicuous  for  the  irregu- 
larity of  their  staining. 

In  cultures  made  upon  two  sets  of  nutrient  agar-agar 
tubes,  differing  only  in  the  fact  that  one  set  contains  glycerin 
to  the  extent  of  6  per  cent.,  while  the  other  set  contains 


486     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

none,  a  noticeable  difference  in  morphology  can  usually 
be  made  out:  while  the  forms  on  the  glycerin-agar-agar 
cultures  are  throughout  small,  and  pretty**  regular  in  size, 
shape,  and  staining,  those  on  the  plain  agar-agar  are  larger, 
stain  less  uniformly,  vary  more  in  shape,  and  when  stained 
by  Loffler's  blue  are  not  so  regularly  marked  by  pale  trans- 
verse lines  that  give  to  them  the  appearance  of  being  made 
up  of  numerous  short  segments. 

Though  the  outline  of  this  organism  is  more  regular 
under  some  circumstances  than  others,  it  is  nevertheless 
always  conspicuous  for  its  manifold  variations  in  shape. 

Growth  on  Serum  Mixture. — The  medium  upon  which 
bacillus  diphtherise  grows  most  rapidly  and  luxuriantly  and 
which  is  best  adapted  for  determining  its  presence  in  diph- 
theritic exudates,  is,  as  has  been  stated,  the  blood-serum 
mixture  of  Loffler.  (See  chapter  on  Media.)  On  the  blood- 
serum  mixture  the  colonies  of  bacillus  diphtherise  grow  so 
much  more  rapidly  than  the  other  organisms  usually  present 
in  secretions  and  exudations  in  the  throat  that  at  the  end  of 
twenty-four  hours  they  are  often  the  only  colonies  that 
attract  attention;  and  if  others  of  similar  size  are  present, 
they  are  generally  of  quite  a  different  aspect.  Its  colonies 
are  large,  round,  elevated,  grayish- white  or  yellowish,  with 
a  center  more  opaque  than  the  slightly  irregular  periphery. 
The  surface  of  the  colony  is  at  first  moist,  but  after  a  day  or 
two  becomes  rather  dry  in  appearance. 

A  blood  serum  tube  studded  with  coalescent  or  scattered 
colonies  .of  this  organism  is  so  characteristic  that  one  familiar 
with  the  appearance  can  anticipate  with  tolerable  certainty 
the  results  of  microscopic  examination. 

Glycerin-agar-agar. — Upon  nutrient  glycerin-agar-agar  the 
colonies  likewise  present  an  appearance  that  readily  may 


BACTERIUM  DIPHTHERIA  487 

be  recognized.  They  are  in  every  way  more  delicate  in 
structure  than  when  on  the  serum  mixture.  They  appear 
at  first,  when  on  the  surface,  as  very  flat,  almost  transparent, 
dry,  non-glistening,  round  points  which  are  not  elevated 
above  the  surface  upon  which  they  are  growing.  When 
slightly  magnified  they  are  seen  to  be  granular,  and  to 
present  an  irregular  central  marking,  which  is  denser  and 
darker  by  transmitted  light  than  the  thin,  delicate  zone 
which  surrounds  it.  As  the  colony  increases  in  size  the  thin 
granular  peripheral  zone  becomes  broader,  is  usually  marked 


FIG.  82 


c* 


Colonies  of  bacterium  diphtherise  on  glycerin-agar-agar.  a,  colonies 
located  in  the  depths  of  the  medium;  6,  colonies  just  breaking  out  upon 
the  surface  of  the  medium;  c,  fully  developed  surface-colony. 

by  ridges  or  cracks,  and  its  periphery  is  notched  or  scalloped. 
(Fig.  80,  c.)  These  colonies  are  always  quite  dry  in  ap- 
pearance. When  deep  down  in  the  agar-agar  they  are 
coarsely  granular.  (Fig.  80,  a.)  They  rarely  exceed  3  mm. 
in  diameter. 

Gelatin. — On  gelatin  the  colonies  develop  much  more 
slowly  than  on  media  that  can  be  retained  at  a  higher  tem- 
perature. They  rarely  present  their  characteristic  appear- 
ances on  gelatin  in  less  than  seventy-two  hours.  They  then 
appear  as  flat,  dry,  translucent  points,  usually  round  in 
outline. 


488      APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

When  magnified  slightly  the  center  is  seen  to  be  more 
dense  than  the  surrounding  zone  or  zones,  for  they  are 
sometimes  marked  by  a  concentric  arrangement  of  zones. 
The  periphery  is  irregularly  notched.  Like  the  colonies 
seen  on  agar-agar,  they  are  granular,  but  are  much  more 
granular  when  seen  in  the  depths  of  the  gelatin  than  when 
on  its  surface.  On  gelatin  the  colonies  rarely  become  very 
large;  usually  they  do  not  exceed  1.5  mm.  in  diameter. 

Bouillon. — In  bouillon  it  usually  grows  in  fine  clumps, 
which  fall  to  the  bottom  of  the  tube,  or  become  deposited 
on  its  sides  without  causing  diffuse  clouding  of  the  bouillon. 
Sometimes  there  are  exceptions  to  this  naked-eye  appear- 
ance; the  bouillon  may  be  diffusely  clouded;  but  if  one 
inspect  it  very  closely,  particularly  if  he  examine  it  micro- 
scopically as  a  hanging  drop,  the  arrangement  in  clumps  will 
always  be  detected,  but  the  clumps  are  so  small  as  not  to 
be  discernible  by  the  unaided  eye. 

In  bouillon  kept  at  a  temperature  of  35°-37°  C.  a  soft, 
whitish  pellicle  often  forms  upon  the  surface. 

The  reaction  of  the  bouillon  frequently  becomes  at  first 
acid,  and  subsequently  again  alkaline,  changes  which  can 
•be  observed  in  cultivations  in  bouillon  to  which  a  little 
rosolic  acid  has  been  added.  This  play  of  reactions  has 
been  attributed  to  the  primary  fermentation  of  the  muscle- 
sugar  often  present  in  the  bouillon.  It  does  not  occur 
when  the  medium  is  free  from  carbohydrates. 

Potato.— On  potato  at  a  temperature  of  35°-37°  C.  its 
growth  after  several  days  is  invisible,  only  a  thin,  dry  glaze 
appearing  at  the  point  at  which  the  potato  was  inoculated. 
Microscopic  examination  of  scrapings  from  the  potato, 
after  twenty-four  hours  at  35°-37°  C.,  reveals  a  decided 
increase  in  the  number  of  individual  organisms  planted. 


BACTERIUM  DIPHTHERIA  489 

Stab-  and  Slant-cultures. — In  stab-  and  slant-cultures  on 
both  gelatin-  and  glycerin-agar-agar  the  surface-growth  is 
seen  to  predominate  over  that  along  the  track  of  the  needle 
in  the  depths  of  the  media. 

Isolated  colonies  on  the  surface  of  either  of  the  media  in 
this  method  of  cultivation  present  the  same  characteristics 
that  have  been  given  for  the  colonies  on  plates. 

The  growth  in  simple  stab-cultures  does  not  extend  later- 
ally very  far  beyond  the  point  at  which  the  needle  entered 
the  medium. 

It  is  a  non-motile  organism. 

It  does  not  form  spores. 

It  is  killed  in  ten  minutes  by  a  temperature  of  58°  C. 

It  grows  at  temperatures  ranging  from  22°  to  37°  C., 
but  most  luxuriantly  at  the  latter  temperature. 

Its  growth  in  the  presence  of  oxygen  is  more  active  than 
when  this  gas  is  excluded. 

Staining. — In  cover-slip  preparations  made  either  from 
the  fauces  of  a  diphtheritic  patient  or  from  a  pure  culture 
of  the  organism  it  is  seen  to  stain  readily  with  the  ordinary 
aniline  dyes.  It  stains  also  by  the  method  of  Gram,  but  the 
best  results  are  obtained  by  the  use  of  Loffler's  alkaline 
methylene-blue  solution;  this  brings  out  the  dark  points 
in  the  protoplasmic  body  of  the  bacilli  and  thus  aids  in  their 
identification.  » 

For  the  purpose  of  demonstrating  the  Loffler  bacillus  in 
sections  of  diphtheritic  membrane,  both  the  Gram  method 
and  the  fibrin  method  of  Weigert  give  excellent  results. 

Pathogenic  Properties. — When  inoculated  subcutaneously 
into  the  bodies  of  susceptible  animals  the  result  is  not  the 
production  of  septicemia,  as  is  seen  to  follow  the  introduc- 
tion into  animals  of  certain  other  organisms  with  which 


490     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

we  shall  have  to  deal,  but  the  bacillus  of  diphtheria  remains 
localized  at  the  point  of  inoculation,  rarely  disseminating 
further  than  the  nearest  lymphatic  glands.  It  develops  at 
the  point  in  the  tissues  at  which  it  is  deposited,  and  during 
its  development  gives  rise  to  changes  in  the  tissues  which 
result  entirely  from  the  absorption  of  poisons  generated  by 
the  bacteria  in  the  course  of  their  development. 

Occasionally  diphtheria  bacilli  may  be  found  in  the 
blood  and  internal  organs  of  individuals  dead  of  the  disease; 
but  all  that  has  been  learned  from  careful  study  of  the 
secondary  manifestations  of  diphtheria  tends  to  the  opinion 
that  they  are  in  no  way  dependent  upon  the  immediate 
presence  of  bacteria,  and  that  the  occasional  appearance 
of  diphtheria  bacteria  in  the  internal  organs  is  in  all  prob- 
ability accidental,  and  usually  unimportant. 

By  special  methods  of  inoculation1  (the  injection  of  fluid 
cultures  into  the  testicles  of  guinea-pigs)  diphtheria  bacilli 
can  be  cawed  to  appear  in  the  omentum;  but  this  is  purely 
an  artificial  manifestation  of  the  disease,  and  one  that  is 
probably  never  encountered  in  the  natural  course  of  events. 
More  rarely  similar  results  follow  upon  subcutaneous 
inoculation. 

If  a  very  minute  portion  of  a  virulent  pure  culture  of 
this  organism  be  introduced  into  the  subcutaneous  tissues 
of  a  guinea-pig  or  kitten,  death  of  the  animal  ensues  in 
from  twenty-four  hours  to  five  days.  The  usual  changes 
are  an  extensive  local  edema,  with  more  or  less  hyperemia 
and  ecchymoses  at  the  site  of  inoculation;  swollen  and  red- 
dened lymphatic  glands;  increased  serous  fluid  in  the  peri- 
toneum, pleura,  and  pericardium;  enlarged  and  hemorrhagic 

1  Abbott  and  Ghriskey,  A  Contribution  to  the  Pathology  of  Experi- 
mental Diphtheria,  The  Johns  Hopkins  Hospital  Bulletin,  No.  30,  April, 
1893. 


BACTERIUM  DIPHTHERIA  491 

adrenal  bodies;  occasionally  slightly  swollen  spleen;  and 
sometimes  fatty  degeneration  in  the  liver,  kidney,  and  myo- 
cardium. In  guinea-pigs,  especially,  the  liver  often  shows 
numerous  macroscopic  dots  and  lines  on  the  surface  and 
penetrating  the  substance  of  the  organ.  They  vary  in  size 
from  a  pin-point  to  a  pin-head,  and  may  be  even  larger. 
They  are  white  and  do  not  project  above  the  surface  of  the 
capsule. 

The  bacteria  are  always  to  be  found  at  the  site  of  inocu- 
lation, most  abundant  in  the  grayish-white,  fibrino-purulent 
exudate.  They  become  fewer  at  a  distance  from  this,  so 
that  the  more  remote  parts  of  the  edematous  tissues  do 
not  contain  them.  They  are  found  not  only  free,  but  con- 
tained in  large  number  in  leukocytes,  some  of  .which  have 
fragmented  nuclei,  or  have  lost  their  nuclei.  The  bacteria 
within  leukocytes,  as  well  as  some  outside,  frequently  stain 
very  faintly  and  irregularly,  and  may  appear  disintegrated 
and  dead. 

Culture  tubes  inoculated  from  the  blood,  spleen,  liver, 
kidneys,  adrenal  bodies,  distant  lymphatic  glands,  and 
serous  transudates,  generally  yield  negative  results;  and 
negative  results  are  also  obtained  when  these  organs  are 
examined  microscopically  for  the  bacteria. 

Microscopic  examination  of  the  tissues  at  the  site  of 
inoculation,  as  well  as  of  the  liver,  spleen,  kidneys,  lymphatic 
glands,  and  elsewhere,  reveals  the  presence  of  localized  foci 
of  cell-death,  characterized  by  a  peculiar  fragmentation  of 
the  nuclei  of  the  cells  of  these  parts. 

This  destruction  of  nuclei  results  in  the  formation  of 
groups  of  irregularly  shaped,  deeply  staining  bodies,  having 
at  times  the  appearance  of  particles  of  dust,  while  again 
they  may  be  much  larger.  Some  of  them  are  tolerably 


492      APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

regular  in  outline,  while  others  are  irregularly  crescentic, 
dumb-bell,  flask-shape,  whetstone-shape,  or  bladder-like 
in  form.  Occasionally  nuclei  having  the  appearance  of 
being  pinched  or  drawn  out  can  be  seen.  At  some  points 
the  fragments  are  grouped  in  isolated  masses,  indicating  the 
location  of  the  nucleus  from  the  destruction  of  which  they 
originated.  'These  particles  always  stain  much  more  in- 
tensely than  do  the  normal  nuclei  of  the  part.1  Oertel 
showed  long  before  bacillus  diphtherise  was  discovered  that 
these  peculiar  alterations  in  cell  nuclei,  both  in  distribution 
and  appearance,  are  characteristic  of  human  diphtheria, 
and  the  demonstration  of  similar  changes  in  animals  inocu- 
lated with  this  organism  is  important  additional  proof  that 
diphtheria  is  caused  by  it. 

By  the  inoculation  of  certain  animals  an  affection  may 
be  produced  in  all  respects  identical  with  diphtheria  as  it 
exists  in  man.  If  one  open  the  trachea  of  a  kitten  and  rub 
upon  the  mucous  membrane  a  small  portion  of  a  pure  culture 
of  this  organism,  the  death  of  the  animal  usually  ensues  in 
from  two  to  four  days.  At  autopsy  the  wound  will  be  found 
covered  with  a  grayish,  adherent,  necrotic,  distinctly  diph- 
theritic layer.  Around  the  wound  the  subcutaneous  tissues 
will  be  edematous.  The  lymphatic  glands  at  the  angle 
of  the  jaws  will  be  swollen  and  reddened.  The  mucous 
membrane  of  the  trachea  at  the  point  upon  which  the  bac- 
teria were  deposited  will  be  covered  with  a  tolerably  firm, 
grayish-white,  loosely  attached  pseudomembrane  in  all 
respects  identical  with  the  croupous  membrane  observed 
in  the  same  situation  in  cases  of  human  diphtheria.  In  the 

1  See  The  Histological  Changes  in  Experimental  Diphtheria,  also  Th.e 
Histological  Lesions  Produced  by  the  Toxalbumin  of  Diphtheria,  by  Welch 
and  Flexner,  Johns  Hopkins  Hospital  Bulletin,  August,  1891,  and  March, 
1892. 


BACTERIUM  DIPHTHERIA  493 

pseudomembrane  and  in  the  edematous  fluid  about  the 
skin-wound  bacillus  diptherise  may  be  found  both  in  cover- 
slips  and  in  cultures. 

From  what  we  have  seen — the  localization  of  the  bacilli 
at  the  point  of  inoculation,  their  absence  from  the  internal 
organs,  and  the  changes  brought  about  in  the  cellular  ele- 
ments of  the  internal  organs — there  is  but  one  interpre- 
tation for  this  process,  viz.,  that  it  is  due  to  the  production 
of  a  soluble  poison  by  the  bacteria  confined  to  the  site  of 
inoculation,  which,  gaining  access  to  the  circulation,  produces 
the  changes  that  we  observe  in  the  tissues  of  the  internal 
viscera. 

This  poison  has  been  isolated  from  cultures  pf  bacillus 
diphtheriae,  and  is  found  to  belong,  not  to  the  crystallizable 
ptomains,  but  to  the  toxins — bodies  which,  in  their  chemical 
composition,  are  analogous  to  the  poison  of  certain  venom- 
ous serpents.  By  the  introduction  of  this  toxin  into  the 
tissues  of  guinea-pigs  and  rabbits  the  same  pathological 
alterations  may  be  produced  that  we  have  seen  to  follow 
inoculation  with  the  bacilli  themselves,  except,  perhaps, 
the  production  of  false  membranes. 

Under  certain  circumstances  with  which  we  are  not 
acquainted  bacillus  diphtherias  becomes  diminished  in 
virulence  or  may  lose  it  entirely,  so  that  it  is  no  longer 
capable  of  producing  death  of  susceptible  animals,  and  may 
cause  only  a  transient  local  reaction  from  which  the  animal 
entirely  recovers.  Sometimes  this  reaction  is  so  slight  as 
to  be  overlooked,  and  again  careful  search  may  fail  to  reveal 
evidence  of  any  reaction  at  all.  These  exhibitions  of  the 
extremes  of  its  pathogenic  properties,  viz.,  death  of  the 
animal,  on  the  one  hand,  and  only  very  slight  local  effects 
on  the  other,  was  at  one  time  thought  to  indicate  the  existence 


494     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

of  two  separate  and  distinct  organisms  that  were  alike  in 
cultural  and  morphological  peculiarities,  but  which  differed 
in  their  disease-producing  power.  Further -studies  on  this 
point  have,  however,  shown  that  genuine  bacillus  diph- 
therise  may  possess  almost  all  grades  of  virulence,  and  that 
absence  of  or  diminution  in  virulence  can  hardly  serve  to 
distinguish  as  separate  species  those  varieties  that  are  other- 
wise alike;  moreover,  the  histological  conditions  found  at 
the  site  of  inoculation  in  animals  that  have  not  succumbed, 
but  in  which  only  the  local  reaction  has  appeared,  are  in 
most  cases  characterized  by  tissue  changes  that  are  identi- 
cal in  kind  though  less  in  degree  to  those  seen  at  autopsy 
in  animals  in  which  inoculation  has  proved  fatal. 

In  the  course  of  their  observations  upon  a  large  number 
of  cases  Roux  and  Yersin  found  that  it  was  not  difficult  to 
detect,  in  the  diphtheritic  deposits  of  a  patient  ill  of  diph- 
theria bacteria  of  identical  cultural  and  morphological 
peculiarities,  but  of  very  different  degrees  of  virulence,  and 
that  with  the  progress  of  the  disease  toward  recovery  the 
less  virulent  varieties  often  became  quite  frequent.1 

There  is,  moreover,  a  mild  form  of  diphtheria,  etiologically 
speaking,  affecting  only  the  mucous  membrane  of  the  nares, 
known  as  membranous  rhinitis,  from  which  it  is  very  com- 
mon to  obtain  cultures  in  all  respects  identical  with  those 
from  typical  diphtheria,  save  for  their  inability  to  kill  sus- 
ceptible animals.  On  inoculation  these  cultures  produce 
only  local  reactions,  but  these  are  characterized  histologi- 
cally  by  the  same  kind  of  tissue-changes  that  follow  inocu- 
lation with  the  fully  virulent  organism. 

1  It  must  not  be  assumed  from  this  that  the  bacteria  lose  their  virulence 
entirely,  or  that  they  all  become  attenuated  with  the  establishment  of  con- 
valescence, for  this  is  contrary  to  what  experience  has  shown  to  be  the  case. 


BACTERIUM  DIPHTHERIA  495 

Clinically,  membranous  rhinitis  is  never  such  an  alarming 
disease  as  is  laryngeal  or  pharyngeal  diphtheria,  and,  as 
stated,  the  organisms  causing  it  are  often  of  a  low  degree  of 
virulence,  though  they  are,  nevertheless,  genuine  diphtheria 
bacteria. 

For  those  organisms  that  are  in  all  respects  identical  with 
the  virulent  bacillus  diphtherise,  save  for  their  inability  to 
kill  guinea-pigs,  the  designation  "  pseudodiphtheritic  bacil- 
lus" is  usually  employed;  but  from  such  observations  as 
those  just  cited  we  are  inclined  to  the  opinion  that  pseudo- 
diphtheritic,  as  applied  to  an  organism  in  all  respects  iden- 
tical with  the  genuine  bacterium,  except  that  it  is  not  fatal 
to  susceptible  animals,  is  a  misnomer,  and  that  it  would 
be  more  nearly  correct  to  designate  this  organism  as  the 
attenuated  or  non-virulent  diphtheritic  bacterium,  reserving 
the  terms  "pseudodiphtheritic"  or  " diphtheroid "  for  that 
organism  or  group  of  organisms  (for  there  are  probably 
several)  that  are  enough  like  the  diphtheria  bacterium  to 
attract  attention,  but  is  distinguishable  from  it  by  certain 
morphological  and  cultural  peculiarities  aside  from  the 
question  of  virulence. 

It  is  a  well-known  fact  that  many  pathogenic  organisms — 
conspicuous  among  these  being  bacterium  pneumoniae, 
micrococcus  aureus,  streptococcus  pyogenes,  and  the  group 
of  so-called  "hemorrhagic  septicemia"  organisms — undergo 
marked  variations  in  their  pathogenic  properties;  and  yet 
these  organisms,  when  found  either  devoid  of  this  peculiarity, 
or  possessing  it  in  a  diminished  degree,  are  not  designated 
as  "pseudo"  forms,  but  simply  as  varieties,  the  virulence  of 
which,  from  various  causes,  has  been  modified. 

It  must  nevertheless  be  admitted  that  in  the  course  of 


496     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

microscopic  examination  of  materials  from  various  sources, 
including  the  pharynx,  one  occasionally  encounters  micro- 
organisms whose  morphology  is  so  like  that  of  the  genuine 
bacterium  diphtheriae  as  to  create  suspicion,  and  yet  they 
are  at  the  same  time  sufficiently  unlike  it  to  make  one  cau- 
tious in  forming  an  opinion  as  to  their  real  nature. 

Variations. — The  pleomorphism  of  bacillus  diphtherise, 
together  with  its  many  irregularities  in  physiological  func- 
tion, render  some  satisfactory  grouping  or  typing,  highly 
desirable.  Numerous  efforts  in  this  direction  have  been 
made  but  none  as  yet  with  entirely  satisfactory  results. 

The  efforts  to  group  the  varieties  of  this  organism  accord- 
ing to  minute  peculiarities  of  form,  structure  and  staining 
peculiarities  and  to  attribute  to  one  such  group  pathogenic 
powers  and  to  another  no  such  power,  involves  far  too 
much  that  is  subjective  'to  be  of  permanent  value,  in  fact 
it  is  in  most  instances  misleading. 

The  grouping  or  typing  according  to  certain  functional 
characteristics,  such  as  zymogenesis,  has  been  of  use,  but  it 
still  leaves  something  to  be  desired. 

The  grouping  in  accordance  with  pathogenic  potency  is 
surrounded  by  too  many  complications  to  be  routinely 
useful. 

The  efforts  to  group  the  large  number  of  varieties  of  this 
species,  though  specific'  agglutinating  reactions,  as  has 
been  done  with  pneumococci,  streptococci,  meningococci 
and  certain  other  organisms,  though  not  far  advanced,  holds 
out,  nevertheless,  much  promise  of  success.  Up  to  the 
present  the  results  of  the  efforts  may  be  summarized  about 
as  follows: 

1.  There  is  apparently  no  constant  relationship  between 
morphology  and  antigenic  power. 


BACTERIUM  DIPHTHERIA  497 

2.  By  immunizing  an  animal  from  any  one  of  a  large 
number  of  strains  of  genuine  bacillus  diphtherise  a  certain 
number  of  the  varieties  in  the  group  will  agglutinate  with 
the  serum  of  that  animal  in  very  high  dilutions;  while  others 
will  either  not  agglutinate  at  all  with  that  serum  or  only  in 
very  low  dilutions. 

3.  These  agglutination  reactions  are  specific  for  the  several 
groups,  i.  e.,  cross-reactions  are  not  observed. 

4.  There  is  a  sharp  distinction  between  the  agglutining 
antigenic  component  and  the  antitoxin  antigenic  component 
in  bacillus  diphtherise. 

5.  The  antitoxin  produced  through  the  use  of  the  several 
groups  are  not  so  sharply  distinguished  from  one  another 
as  are  the  agglutinins,  though  they  manifest  specific  rela- 
tionship to  their  homologous  antigens. 

The  bearing  of  all  this  on  the  recognition  of  bacillus 
diphtheria  and  on  the  production  of  antitoxin  is  obvious.1 

Bacterium  Pseudodiphtheriticum. — For  a  long  time  bac- 
terium pseudodiphtheriticum  was  looked  upon  as  being 
entirely  harmless,  and  the  only  particular  in  which  it  was 
regarded  as  being  of  importance  was  in  the  fact  that  it  was 
likely  to  be  mistaken  for  bacterium  diphtherise.  The  wide 
dissemination  of  this  class  of  organisms  and  the  demon- 
stration of  pathogenic  effects  in  isolated  instances  has  led 
to  the  more  systematic  study  of  members  of  this  group  of 
organisms. 

Bacterium  pseudodiphtheriticum,  as  found  under  different 
conditions,  varies  markedly  in  its  morphologic  and  biologic 
characters.  Some  of  the  varieties  have  definite  chromogenic 

1  See  Langer,  Die  Agglutination  der  Diphtherie  bacillen,   Centralbl.  f. 
Bact.,  Abt.  I,  Originale,  1916,  vol.  Ixxviii,  p.  117.     Havens,  Biologic  Studies 
of  the  Diphtheria  Bacillus,  Jour.  Infect.  Dis.,  1920,  vol.  xxvi,  p.  38$,     Addi- 
tional literature  given  .in  these  papers. 
32 


498     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

properties,  producing  various  shades  of  yellow-  and  orange- 
colored  pigment,  while  others  grow  with  a  pink  color. 

The  occurrence  of  bacterium  pseudodiphthetiticum  in 
pure  culture  in  superficial  abrasions  showing  a  slight  ten- 
dency to  suppuration;  the  fact  that  these  organisms,  when 
injected  into  the  peritoneal  cavity  of  guinea-pigs,  produce 
purulent  peritonitis;  that  such  organisms  are  frequently 
encountered  in  vaccine  virus  and  in  the  pus  of  vaccination 
wounds;  and  that  frequently  in  cases  of  mastitis  in  cows 
such  organisms  occur  in  large  numbers  in  pure  culture  has 
led  to  the  supposition  that  this  group  of  organisms  was 
probably  responsible  for  suppurations  occurring  under 
certain  special  conditions.  With  these  facts  in  mind  speci- 
mens of  pus  were  derived  from  thirty  cases  with  suppurating 
wounds  in  the  University  of  Pennsylvania  Hospital,  and 
careful  bacteriological  examination  of  these  specimens 
showed  the  presence  of  bacterium  pseudodiphtheriticum  in 
43  per  cent,  of  the  cases.  These  organisms  were  always 
found  in  conjunction  with  one  or  more  of  the  group  of  pyo- 
genic  organisms,  and  it  is  impossible  to  state  how  much 
of  the  effect  was  due  to  any  one  of  the  organisms  present. 
It  seems  probable,  however,  in  the  light  of  what  has  been 
said,  that  these  bacteria  were  present  not  merely  as  acci- 
dental invaders,  but  that  in  some  way  they  contributed 
toward  the  results. 

The  fact  that  some  of  the  organisms  isolated  from  the 
pus,  when  inoculated  into  the  peritoneal  cavity  of  guinea- 
pigs,  show  distinct  pyogenic  properties  gives  strong  sup- 
port to  the  opinion  that  this  group  is  of  greater  importance 
than  was  heretofore  supposed.  Repeated  passage  through 
guinea-pigs  serves  to  so  increase  the  pathogenic  properties 
of  these  organisms  that  they  cause  theMeath  of  the  animal 


BACTERIUM  XEROSIS  499 

in  less  than  twenty-four  hours  with  marked  inflammatory 
reaction  affecting  the  peritoneum  as  well  as  the  abdominal 
organs. 

The  morphologic  and  biologic  characters  of  some  members 
of  the  group  of  bacterium  pseudodiphtheriticum  are  sug- 
gestive of  those  of  bacterium  diphtherise.  Other  members 
of  the  group,  however,  are  readily  differentiated  from  bac- 
terium diphtherias  by  either  the  morphologic  or  the  biologic 
characters,  or  by  both.  Many  of  the  members  of  the  group 
produce  very  little  acid  when  grown  in  carbohydrate  media, 
and  the  slight  degree  of  acidity  produced  is  frequently 
obliterated  by  a  marked  degree  of  subsequent  alkali 
production.  This  fact  is  of  special  value  in  the  differentia- 
tion from  bacterium  diphtherise. 

BACTERIUM  XEROSIS  (NEISSER  AND  KUSCHBERT), 
MIGULA,  1900. 

SYNONYM:    Bacillus  xerosis,  Neisser  and  Kuschbert,  1883. 

Another  organism  which  is  also  related  in  its  morphologic 
and  biologic  characters  to  bacterium  diphtherise  is  bacterium 
xerosis,  first  encountered  by  Kuschbert  and  Neisser  in  xerosis 
of  the  conjunctiva,  and  which  has  since  been  found  on  the 
conjunctiva  by  a  number  of  investigators,  in  various  diseases 
as  well  as  in  health. 

The  xerosis  bacteria  are  less  likely  to  be  mistaken  for 
bacterium  diphtherias  because  they  are  somewhat  smaller 
and  have  less  tendency  to  show  multiple  striations.  Usually 
they  stain  deeply  at  the  poles  with  only  one  clear  unstained 
band  in  the  center.  It  is  only  occasionally  that  a  few  striated 
organisms  are  encountered  in  a  culture. 


500     APPLICATION  OF  METHODS  QF  BACTERIOLOGY 

Biologically  bacterium  xerosis  is  readily  differentiated 
from  bacterium  diphtherise  because  of  the  scant  growth 
that  takes  place  on  the  ordinary  culture  media.  On  agar- 
agar  the  growth  appears  as  small  transparent  colonies  which 
have  little  tendency  to  coalesce.  On  gelatin  the  growth 
is  slow,  and  frequently  shows  as  minute,  isolated  colonies 
along  the  needle  track.  In  litmus-milk  a  slight  degree  of 
acidity  is  produced.  In  bouillon  the  growth  is  so  slight  as 
to  leave  the  medium  practically  unaltered.  The  growth 
on  potato  is  slight  and  invisible. 

Differentiation  of  Members  of  the  Group. — Knapp1  claims 
that  a  positive  differentiation  of  the  organisms  may  be 
made  by  merely  inoculating  the  Hiss  media  containing  dex- 
trin and  saccharose.  If  the  dextrin  is  alone  fermented,  the 
organism  is  bacterium  diphtheriae,  if  only  the  saccharose  is 
fermented,  the  organism  is  bacterium  xerosis,  and  if  neither 
of  these  carbohydrates  is  fermented,  the  organism  is  bac- 
terium pseudodiphtheriticum. 

Through  the  suggestion  of  Neisser2  we  are  assisted  in 
differentiating  between  bacillus  diphtherise  and  the  confusing 
forms.  He  has  found  that  by  the  use  of  a  particular  staining 
method  the  appearance  of  bacterium  diphtherise  is  charac- 
teristic. His  differential  method  comprehends  the  following 
manipulations:  the  culture  to  be  tested  should  be  grown 
upon  Loffler's  blood  serum  mixture  solidified  at  100°  C.; 
it  should  develop  at  a  temperature  not  lower  than  34°  C. 
and  not  higher  than  36°  C.;  and  it  should  not  be  younger 
than  nine  and  not  older  than  twenty-four  hours.  A  cover- 
glass  preparation  made  from  such  a  culture  is  stained  as 
follows : 

1  Jour.  Med.  Research,  1904,  xii,  475. 

2  Zeitschrift  fur  Hygiene  und  Infektionskrankheiten,  1897,  Bd.  xxiv. 


BACTERIUM  XEROSIS  501 

(a)  It  is  subjected  to  the  following  mixture  for  from  one 
to  three  seconds: 

Methylene-blue  (Griibler's) 1  gram 

Alcohol  (96  per  cent.)          .      .*»,./.      •  >t  •      •      •     20  c.c. 

When  dissolved,  mix  with 

Acetic  acid '.'..,.  \..    .      .      .      .50  c.c. 

Distilled  water ..'.'.      .      .  950  c.c. 

(6)  After  thoroughly  rinsing  in  water,  it  is  stained  for 
from  three  to  five  seconds  in  vesuvin  (Bismarck-brown), 
2  grams,  dissolved  in  1  liter  of  boiling  distilled  water, 
filtered,  ond  allowed  to  cool.  It  is  again  rinsed  in  water 
and  examined  as  a  water-mount,  or  it  may  be  dried  and 
mounted  in  balsam. 

When  so  treated  the  diphtheria  bacterium  appears  as 
faintly  stained  brown  rods,  in  which  from  one  to  three 
dark-blue  granules  are  to  be  observed.  The  dark  granules 
are  at  one  or  both  poles  of  the  cell,  are  more  or  less  oval, 
and  usually  seem  to  bulge  a  little  beyond  the  contour  of  the 
bacterium  in  which  they  are  located.  (See  Fig.  83.)  From 
Neisser's  observations  and  those  of  others,1  as  well  as  from 
personal  experience,  it  seems  safe  in  the  vast  majority  of 
cases  to  regard  all  bacteria  that  do  not  stain  in  the  way 
described  as  distinct  from  bacterium  diphtherise. 

Blumenthal  and  Lipskerow2  decide  that  the  differential 
method  which  yields  the  most  satisfactory  results  consists 
in  the  fixation  of  the  preparation  for  from  one-half  to  two 
minutes  in  the  following  solutions:  Pyoktanin  (Merck) 
0.25  grams,  acetic  acid  (5  per  cent.)  100  c.c.;  washing  in 

1  Frankel,  Berliner  klin.  Wochenschrift,  1897,  No.  50.    Bergey,  Publica- 
tions of  the  University  of  Pennsylvania,  New  Series,  1898,  No.  4. 

2  Centralblatt  f.  Bacteriologie,  Bd.  xxxviii,  p.  359. 


502     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

water  and  counterstaining  with  a  1  to  1000  solution  of 
vesuvin  for  one-half  minute.  By  this  method  the  polar 
granules  of  bacterium  diphtherias  are  stained  bluish  black, 
are  large,  and  may  be  seen  in  almost  all  of  the  organisms. 
The  contour  of  the  darkly  stained  bacterium  diphtherias 
is  sharply  defined,  and  it  is  very  easily  differentiated  from 
any  other  organisms  that  may  be  present  in  the  preparation. 

FIG.  83 


Bacterium  diphtherias,  stained  by  Neisser's  method. 


NOTE. — Prepare  cover-slip  preparations  from  the  mouth- 
cavities  of  healthy  individuals  and  from  those  having  decayed 
teeth.  Do  they  correspond  in  any  way  with  those  made 
from  diphtheria?  Do  the  same  with  different  forms  of 
sore-throat.  Do  the  peculiarities  of  any  of  the  organisms 
suggest  those  of  bacterium  diphtherise?  Wherein  is  the 
difference  ? 

In  cultures  and  cover-slips  made  from  both  diphtheritic 


BACTERIUM  XEROSIS  503 

and  from  innocent  sore-throats  are  any  organisms  almost 
constantly  present?  Which  are  they,  and  what  are  their 
characteristics? 

Which  are  the  predominating  organisms  in  the  anginas 
of  scarlet  fever? 

Do  these  organisms  simulate,  in  their  cultural  and  mor- 
phological peculiarities,  any  of  the  different  species  with 
which  you  have  been  working? 

Do  the  diphtheria  organisms  disappear  from  the  throat 
with  the  disappearance  of  the  membrane?  How  long  do 
they  persist?  When  obtained  from  the  throats  of  convales- 
cents are  they  still  pathogenic  for  guinea-pigs  ? 

Prepare  a  bouillon  culture  of  virulent  bacillus  diphtherise; 
after  it  has  been  growing  for  thirty-six  hours  at  37°-38°  C. 
inoculate  a  guinea-pig  subcutaneously  with  about  0.1  c.c. 
of  it.  If  the  animal  dies,  note  carefully  the  findings  at 
autopsy,  especially  the  distribution  of  the  bacilli.  Now  add 
to  this  culture  sufficient  pure  carbolic  acid  or  trikresol  to 
kill  all  bacteria  in  it,  and  inject  under  the  skin  of  another 
guinea-pig  varying  amounts  of  the  culture  so  treated, 
beginning  with  0.05  c.c. ;  determine  the  minimum  fatal  dose, 
and  note  in  which  respects  the  postmortem  findings  simulate 
and  in  which  they  differ  from  those  of  the  first  animal. 
Should  any  of  the  animals  survive  the  injections  of  the 
disinfected  culture,  note  carefully  their  condition  from  day 
to  day,  particularly  any  fluctuations  in  weight.  When 
they  have  quite  recovered  inoculate  them  with  living, 
virulent  diphtheria  organisms.  Do  the  results  correspond 
with  those  obtained  with  guinea-pigs  that  have  never  been 
treated  at  all?  Explain  the  results. 

Diphtheria  Antitoxin. — As  stated  above,  the  growth  of 
bacterium  diphtherise  is  accompanied  by  the  elaboration 


504     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

of  a  poison  of  remarkable  toxicity  that  is  accountable  for 
the  constitutional  symptoms  and  pathological  lesions  by 
which  the  disease  is  characterized.  If  by  appropriate 
methods  this  poison  (toxin)  be  separated  from  the  bacteria 
by  which  it  was  formed,  it  is  capable,  when  injected  into 
susceptible  animals,  of  causing  death  and  practically  all  the 
lesions  that  accompany  the  disease  when  due  to  the  invasion 
of  the  living  bacteria.  If,  on  the  contrary,  the  dose  of  poison 
be  so  adjusted  as  to  cause  only  temporary  inconvenience 
and  not  endanger  life,  and  this  dose  be  injected  repeatedly, 
gradually  increasing  in  size  as  the  animal  is  able  to  bear 
it,  after  a  while  a  marked  tolerance  is  established,  so  that 
the  animal  may  be  given  many  times  the  amount  of  the 
toxin  that  would  otherwise  prove  fatal — i.  e.,  many  times 
the  lethal  dose  for  an  animal  that  had  not  acquired  such  a 
tolerance. 

If  blood  be  now  drawn  from  the  animal  that  has  become 
habituated,  so  to  speak,  to  the  diphtheria  toxin,  and  the 
serum  collected  from  it,  we  discover  several  important 
facts,  viz.: 

That  this  serum  when  mixed  with  the  previously  deter- 
mined lethal  dose  of  the  toxin  in  a  test-tube  will  either 
neutralize  its  toxicity  or  greatly  reduce  it,  according  to  the 
amount  of  serum  used. 

That  if  we  inject  into  an  animal  the  determined  fatal 
dose  of  the  toxin,  and  immediately  afterward  inject  a  quan- 
tity of  the  .serum,  either  the  animal  will  not  die  or  the  death 
will  be  more  or  less  delayed,  according  to  the  amount  of 
serum  employed. 

That  if  a  susceptible  animal  be  inoculated  with  a  living 
culture  of  virulent  bacterium  diphtheriae,  its  life  may  be 
saved,  or  its  death  postponed,  by  the  subsequent  injection 


BACTERIUM  XEROSIS  505 

of  the  serum;  the  result  depending  upon  the  amount  of 
serum  used  and  the  lapse  of  time  between  inoculation  with 
the  bacteria  and  injection  of  the  serum. 

And,  finally,  that  although  this  serum  has  such  a  marked 
effect  upon  the  toxins  of  bacterium  diphtherise  in  a  test- 
tube  or  in  the  animal,  and  so  striking  an  influence  upon 
the  course  of  infection  with  the  living  organisms  in  the 
animal,  it  has  little  or  no  effect  upon  the  living  bacteria 
either  in  a  test-tube  or  at  the  site  of  inoculation  in  the 
living  animal  body. 

This  serum  with  which  we  have  been  experimenting  is 
the  so-called  "diphtheria  antitoxin"  or  " antidiphtheritic 
serum." 

For  practical  purposes,  it  is  obtained  from  horses,  the 
animals  being  treated  with  gradually  increasing  doses  of 
diphtheria  toxin  until  they  are  able  to  withstand  enormous 
multiples  of  the  ordinarily  fatal  dose.  When  this  point  is 
reached,  the  protective  body — the  antitoxin — is  present  in 
the  blood  in  such  large  quantities  that  the  serum  may  be 
successfully  employed  in  the  treatment  of  diphtheria  in 
human  beings — i.  e.,  as  an  antidote  to  the  diphtheria  toxin 
that  is  produced  by  the  growing  bacteria  in  the  throat,  or 
elsewhere,  and  distributed  through  the  body  by  the  cir- 
culating fluids. 

The  Standardization  of  Diphtheria  Antitoxin. — The  value 
of  diphtheria  antitoxin  may  be  determined  according  to 
several  different  standards.  Those  that  are  best  known 
have  been  proposed  by  Behring  and  by  Ehrlich. 

1.  Behring' s  Method. — He  designates  as  a  "normal"  poison 
a  toxin  of  which  0.01  c.c.  suffices  to  kill  a  guinea-pig  weigh- 
ing 250  grams  in  four  days.  Of  such  a  normal  diphtheria 
toxin  1  c.c.  will  be  sufficient  to  kill  100  guinea-pigs  weigh- 


506     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

ing  250  grams  each,  or  25,000  grams  in  weight  of  guinea- 
pigs. 

The  quantity  of  antitoxin  that  is  required  to  just  protect 
25,000  grams  weight  of  guinea-pigs  from  the  minimum  fatal 
dose  of  the  toxin  is  called  one  immunizing  unit.  If  an 
immune  serum  contains  in  1  c.c.  one  immunizing  unit,  it 
represents  a  "normal"  antitoxin. 

To  determine  the  strength  of  an  immune  serum,  1  c.c.  of 
normal  toxin  is  mixed  with  increasing  quantities  of  the 
serum,  and  these  mixtures  are  injected  subcutaneously  into 
guinea-pigs;  the  quantity  of  the  serum  which  suffices  to 
neutralize  that  amount  of  normal  toxin — i.  e.,  that  keeps  the 
animal  alive  for  four  days  or  longer — contains  one  immunizing 
unit. 

2.  Ehrlich's  Method. — Ehrlich  introduced  the  use  of  a 
standard  diphtheria  antitoxin  in  a  dry  state  which  contains 
1700  immunizing  units  in  each  gram.  This  standard  anti- 
toxin, distributed  by  the  Institute  for  testing  serum  at 
Frankfort-on-the-Main,  is  now  being  used  in  a  great  many 
places  for  the  standardization  of  diphtheria  antitoxin.  A 
test  toxin  is  prepared,  corresponding  to  this  standard  anti- 
toxin, and  with  this  toxin  the  strength  of  the  unknown  serum 
is  titrated. 

If,  for  instance,  the  test  toxin  is  of  such  a  strength  that 
0.003  c.c.  represents  the  minimum  fatal  dose  for  a  guinea- 
pig  of  250  grams,  then  0.3  c.c.  would  represent  100  times 
the  minimum  fatal  dose  of  toxin,  and,  according  to  Ehrlich's 
standard,  an  immunity  unit  is  that  amount  of  antitoxic 
serum  which  will  neutralize  100  times  the  minimum  fatal 
dose  of  toxin.  In  performing  the  test  to  estimate  the 
strength  of  an  antitoxic  serum,  the  antitoxin  is  diluted 
with  sterile  water  in  varying  proportions,  and  a  series  of 


BACTERIUM  XEROSIS  507 

guinea-pigs  are  injected  with  mixtures  of  100  times  the 
minimum  fatal  dose  of  the  toxin  and  varying  quantities  of 
the  diluted  antitoxic  serum.  For  this  purpose  guinea-pigs 
of  approximately  250  grams  weight  are  employed.  If,  for 
instance,  a  guinea-pig  receiving  100  times  the  minimum  fatal 
dose  of  toxin,  and  0.1  c.c.  of  the  diluted  antitoxic  serum, 
survives  for  four  days,  then  0.1  c.c.  of  the  serum  represents 
an  immunity  unit  of  antitoxin. 

An  antitoxic  serum  of  this  strength,  therefore,  contains 
10  times  the  normal  amount  of  antitoxin,  because  it  con- 
tains the  immunity  unit  in  only  0.1  c.c.;  a  normal  anti- 
toxin being  one  in  which  an  immunity  unit  is  contained  in 
one  cubic  centimeter.  Antitoxic  serums  are  frequently  of 
such  high  degree  of  potency  that  they  contain  from  800 
to  1000  immunity  units  in  each  cubic  centimeter. 


CHAPTER  XXIV. 


Typhoid  Fever — Study  of  the  Organism  Concerned  in  its  Production — 
Its  Morphological,  Cultural,  and  Pathogenic  Properties — Bacillus  Coli 
— Bacillus  Paratyphosus — Its  Resemblance  to  Bacillus  Typhosus. 


BACILLUS    TYPHOSUS. 

THE  organism  seen  in  the  cadavers  of  typhoid  subjects 
by  Eberth  (1880-81),  and  subsequently  isolated  in  pure 
culture  and  described  by  Gaffky  (1884),  is  generally  recog- 
nized as  the  exciting  factor  of  typhoid  fever.  It  may  be 
described  as  follows: 


FIG.  84 


FIG.  85 


Bacillus    typhosus,   from   cultures 
twenty-four  hours  old,  on  agar-agar, 


Bacillus  typhosus,  showing 
flagella  stained  by  Loffler's 
method. 


Morphology. — It  -is  a  bacillus  about  three  times  as  long 
as  broad,  with  rounded  ends.  It  may  appear  at  one  time 
as  very  short  ovals,  at  another  time  as  long  threads,  and 
both  forms  may  occur  together.  Its  breadth  remains  toler- 

(508) 


BACILLUS  TYPHOSUS  509 

ably  constant.  Its  morphology  presents  little  that  will 
aid  in  its  identification.  (See  Fig.  84.)  It  is  actively  motile, 
and  when  stained  by  special  methods,  is  seen  to  possess  very 
delicate  locomotive  organs  in  the  form  of  fine,  hair-like 
flagella,  attached  in  large  numbers  to  all  parts  of  its  surface. 
(See  Fig.  85.)  These  flagella  are  not  seen  in  unstained 
preparations,  nor  are  they  rendered  visible  by  ordinary 
methods  of  staining.  (See  methods  for  staining  flagella.) 

Owing  to  a  tendency  to  retraction  of  its  protoplasm  from 
the  cell-envelope  and  the  consequent  production  of  vacuoles 
in  the  bacilli,  the  staining  of  this  organism  is  frequently 

FIG.  86 


Diagrammatic  representation  of  retraction  of  protoplasm,  with  production 
of  pale  points,  in  bacillus  typhosus. 


more  or  less  irregular.  At  some  points  in  a  single  cell  marked 
differences  in  the  intensity  of  the  staining  will  be  seen,  and 
here  and  there  areas  quite  free  from  color  can  commonly 
be  detected.  These  colorless  portions  are  often  so  sharply 
defined  that  they  look  as  if  they  had  been  punched  out 
with  a  sharp  instrument.  (See  Fig.  86.) 

It  does  not  form  spores. 

Gelatin  Plates. — Its  growth,  when  seen  in  the  depths  of 
the  medium,  presents  nothing  characteristic,  appearing 
simply  as  round  or  oval,  finely  granular  points.  On  the 
surface  it  develops  as  very  superficial,  blue-white  colonies, 
with  irregular  borders.  They  are  a  little  denser  at  the 


510     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

center  than  at  the  periphery.  When  magnified,  the  colonies 
present  wrinkles  or  folds,  which  give  to  them,  in  miniature, 
the  appearance  seen  in  relief  maps  (Fig.  87).  These  colonies 
have  sometimes  the  appearance  of  flattened  pellicles  of  glass- 
wool,  and  usually  a  pearl-like  luster. 

Agar-agar. — On  agar-agar  the  colonies  present  nothing 
typical. 

Stab-cultures. — In  stab-cultures  the  growth  is  mostly  on 
the  surface,  there  being  only  a  very  limited  development 
down  the  track  made  by  the  needle.  The  surface  growth 
has  the  same  appearance  in  general  as  that  given  for  the 
colonies. 

FIG.  87 


Colony  of  bacillus  typhosus  on  gelatin, 

Potato. — The  growth  on  potato  is  usually  described  as 
luxuriant  but  invisible,  making  its  presence  evident  only 
by  the  production  of  a  slight  increase  of  moisture  at  the 
inoculated  point,  and  by  a  limited  resistance  offered  to  a 
needle  when  it  is  scraped  across  the  track  of  growth.  While 
this  is  so  in  many  cases,  yet  it  cannot  be  considered  as 
invariable,  for  at  times  this  organism  develops  more  or  less 
visibly  on  potato. 

Potato-gelatin. — The  growth  is  similar  to  that  upon 
ordinary  nutrient  gelatin. 

Milk. — It  does  not  cause  coagulation  when  grown  in 
sterilized  milk. 


BACILLUS  TYPHOSUS  511 

Bouillon. — It  causes  uniform  clouding  of  the  bouillon  and 
brings  about  a  slightly  acid  reaction. 

Indol  Formation. — It  is  customary  to  regard  this  organism 
as  devoid  of  the  power  to  form  indol;  in  fact,  this  has  hitherto 
been  considered  one  of  its  important  differential  peculi- 
arities, and  by  the  usual  methods  of  cultivation  and  test- 
ing the  indol  reaction  is  not  observed  in  cultures.  It  has 
been  shown,  however,  by  Peckham,  that  by  repeated 
transplantation,  at  short  intervals,  into  either  Dunham's 
peptone  solution,  or,  preferably,  a  freshly  prepared  alkali- 
tryptone  solution,  made  from  tryptonized  beef-muscle,  that 
the  indol-producing  function  may  be  induced  in  the  genuine 
typhoid  bacillus  obtained  directly  from  the  spleens  of 
typhoid  cadavers.1 

It  does  not  produce  gaseous  fermentation.  On  lactose- 
litmus-agar-agar  it  grows  as  pale-blue  colonies,  causing  no 
reddening  of  the  surrounding  medium;  though  if  glucose 
be  substituted  for  lactose,  both  the  colonies  and  the  sur- 
rounding medium  may  become  red.  In  the  fermentation- 
tube,  in  glucose  or  lactose  bouillon,  no  evolution  of  gas 
as  a  result  of  fermentation  occurs. 

It  grows  at  any  temperature  between  20°  and  38°  C., 
though  more  favorably  at  the  latter  point.  It  is  very  sen- 
sitive to  high  temperatures,  being  killed  by  an  exposure  of 
ten  minutes  to  60°  C.,  and  in  a  much  shorter  time  to  slightly 
higher  temperatures. 

It  does  not  liquefy  gelatin. 

It  grows  both  with  and  without  oxygen. 

It  does  not  grow  rapidly. 

1  A.  W.  Peckham,  The  Influence  of  Environment  Upon  the  Biological 
Functions  of  the  Colon  Group  of  Bacilli,  Journal  of  Experimental  Medi- 
cine, 1897,  vol.  ii. 


512     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

Presence  in  Tissues. — In  patients  suffering  from  typhoid 
fever  the  organism  has  been  found  during  life  by  the  appli- 
cation of  appropriate  culture  methods  in  the  blood,  urine, 
and  feces,  and  at  autopsies  in  the  tissues  of  the  spleen,  liver, 
kidneys,  intestinal  lymphatic  glands,  and  intestines.  It  is 
not  easy  to  demonstrate  this  organism  in  tissues  unless  it  is 
present  in  large  numbers.  The  manipulations  to  which 
the  sections  are  subjected  in  being  mounted  often  rob  the 
bacilli  of  their  stain,  and  render  them  invisible,  or  nearly  so. 
If,  however,  sections  be  stained  in  the  carbol-fuchsin  or  the 
alkaline  methylene-blue  solution,  either  at  the  ordinary 
temperature  of  the  room  or  at  a  higher  temperature  (40°  to 
45°  C.),  then  washed  in  absolute  alcohol,  and  cleared  in 
xylol1  and  mounted  in  xylol  balsam,  the  bacilli  (particu- 
larly if  the  tissues  be  the  liver  and  spleen)  can  readily  be 
detected,  massed  together  in  clumps. 

In  searching  for  the  typhoid  bacilli  in  tissues  this  peculiar 
disposition  in  clumps  must  always  be  borne  in  mind,  other- 
wise much  labor  will  be  expended  in  vain.  In  tissues  the 
typhoid  bacilli  are  not  scattered  about  as  are  the  organ- 
isms in  certain  other  conditions — septicemia,  for  instance; 
they  are  not  regularly  distributed  along  the  course  of  the 
lymphatics  or  capillaries,  but  appear  in  small  masses  through 
the  organs,  and  it  is  for  these  agglutinations  that  one  should 
search.  This  peculiar  clumping  of  the  typhoid  bacilli  in 
the  tissues  cannot  be  satisfactorily  explained,  unless  it  be 
due  to  the  specific  agglutinating  influence  that  typhoid 
blood  has  upon  the  typhoid  bacillus,  a  phenomenon  that 
is  readily  demonstrable  in  the  test-tube  or  under  the  micro- 
scope. In  other  words,  may  it  not  be  simply  the  result  of  an 
intracapillary  "Widal  reaction"?  (See  Widal  Reaction.) 

1  Do  not  clarify  with  oil  of  cloves.    It  is  too  active  as  a  decolorizer. 


BACILLUS  TYPHOSUS  513 

When  the  section  is  prepared  for  examination,  if  it  be  gone 
over  with  a  low-power  objective,  one  will  notice  at  irregular 
intervals  little  masses  that  look  in  every  respect  like  par- 
ticles of  staining-matter  which  have  been  precipitated  upon 
the  section  at  that  point.  When  these  masses  are  examined 
with  a  higher  power  objective  they  will  be  found  to  consist 
of  small  ovals  or  short  rods  so  closely  packed  that  the  indi- 
viduals composing  the  clump  can  often  be  seen  only  at  the 
extreme  periphery  of  the  mass.  This  is  the  characteristic 
appearance  of  the  typhoid  organism  in  tissues,  to  which 
allusion  has  just  been  made.  The  little  masses  are  usually 
in  the  neighborhood  of  a  capillary. 

Isolation  of  Bacillus  Typhosus  from  Cadavers. — The  spleen 
of  a  patient  dead  of  typhoid  fever  is  the  most  reliable  source 
from  which  to  obtain  cultures  of  the  typhoid  bacillus  for 
study.  But  it  must  always  be  remembered  that  the  same 
channels  through  which  the  typhoid  bacillus  gains  access 
to  this  viscus  are  likewise  open  to  other  organisms  present 
in  the  intestines,  and  for  this  reason  bacillus  coli,  a  normal 
inhabitant  of  the  colon,  may  also  be  found  in  this  locality. 

Result  of  Inoculation  into  Lower  Animals. — A  great  many 
experiments  have  been  made  in  a  variety  of  ways  with  the 
view  of  reproducing  the  pathological  conditions  of  this 
disease,  as  seen  in  man,  in  the  tissues  of  lower  animals,  but 
with  practically  no  success.  From  the  time  of  its  discovery 
up  to  within  a  comparatively  recent  date  there  was  an  almost 
continuous  controversy  concerning  the  infective  properties 
of  bacillus  typhosus  for  animals.  By  some  it  was  held  that 
the  effects  of  its  introduction  into  animals  were  manifestly 
of  toxic1  origin,' while  others  regarded  them  as  evidences  of 

1  Toxic — poisonous  results  not  necessarily  accompanied  by  the  growth 
of  organisms  throughout  the  tissues. 
33 


514     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

genuine  infection.1  These  diversities  of  opinion  are  hardly 
surprising  when  we  remember  that  animals  never  suffer 
naturally  from  typhoid  fever,  and  therefore  offer  many 
obstacles  to  its  faithful  reproduction,  and  that  the  vigor  of 
this  organism  when  cultivated  from  various  sources  is  liable 
to  a  wide  range  of  fluctuation.  Numerous  investigations 
lead  to  the  belief  that  the  poison  peculiar  to  this  organism 
is  so  intimately  bound  up  with  its  protoplasmic  structure 
as  to  make  its  separation  difficult,  if  not  impossible.  How- 
ever, by  the  use  of  dead  cultures  (i.  e.,  cultures  of  well 
developed  organisms  destroyed  by  heat)  results  are  obtained 
that  leave  no  doubt  that  the  clinical  symptoms  and  patho- 
logical changes  seen  in  man  and  in  animals  under  experiment 
are  referable  to  a  specific  intoxication,  and,  as  a  rule,  the 
only  effects  that  follow  the  introduction  of  this  organism 
into  animals  are  referable  to  the  intoxicating  action  of  the 
materials  used.  In  fact,  the  results  of  modern  investigations 
have  placed  bacillus  typhosus  in  the  category  of  endotoxin 
producers,  and  through  the  use  of  the  toxins  (not  pure,  but 
mixed  with  other  substances  in  the  culture  media)  produced 
by  it  animals  have  been  rendered  immune  from  otherwise 
fatal  doses  of  highly  toxic  cultures.  The  serum  of  such 
animals  has  also  been  shown  to  possess  a  certain  degree  of 
immunizing  power.2 

Because  of  the  variations  in  the  morphology  and  physiology 
of  this  organism,  and  because  of  the  difficulty  experienced 
in  efforts  to  reproduce  in  lower  animals  the  condition 
found  in  the  human  subject,  our  knowledge  of  typhoid 
fever,  though  fairly  accurate  in  many  respects,  is,  never- 

1  Infective  or  septic — poisoning  of  the  tissues  as  a  result  of  the  growth 
of  bacteria  within  them. 

2  Pfeiffer  and  Kolle,  Zeitschrift  fur  Hygiene  und  Infektionskrankheiten 
1896,  Bd.  xxi,  S.  208. 


BACILLUS  TYPHOSUS  515 

theless,  in  certain  essential  details  relating  to  its  causation, 
very  far  from  satisfying. 

A  number  of  other  organisms  appear  botanically  to  be 
closely  related  to  the  typhoid  bacillus,  and  under  the  avail- 
able culture  methods  for  studying  them  they  so  closely 
simulate  it  that  the  difficulty  of  identifying  this  organism 
is  sometimes  very  great.  In  addition  the  variability  con- 
stantly seen  in  pure  cultures  of  the  typhoid  bacillus  itself 
in  no  way  renders  the  task  more  simple. 

For  example,  the  morphology  of  the  typhoid  bacillus 
is  conspicuously  inconstant;  its  growth  on  potato  which  was 
formerly  considered  unique,  may,  with  the  same  stock,  at 
one  time  be  the  typical  invisible  development,  at  another 
it  is  easily  to  be  seen  with  the  naked  eye;  and  the  change  of 
reaction  which  it  is  said  to  produce  in  bouillon  is  sometimes 
much  more  intense  than  at  others.  The  indol-producing 
function,  hitherto  regarded  as  absent  from  this  organism, 
is  now  known  to  be  occasionally  demonstrable  by  ordinary 
methods,  and  frequently  by  special  methods  of  cultivation 
(Peckham,  /.  c.).  The  only  properties  exhibited  by  it  under 
the  usual  conditions  of  cultivation  that  may  be  said  to  be 
constant  are  its  motility;  its  inability  to  cause  gaseous  fer- 
mentation of  glucose,  lactose,  or  saccharose;  its  incapacity 
for  coagulating  milk;  and  its  growth  on  gelatin  plates 
but  there  are  other  bacilli  which  possess  these  same  character- 
istics to  a  degree  that  renders  their  differentiation  from 
the  typhoid  organism  often  a  matter  that  requires  the  careful 
application  of  all  the  different  tests. 

The  Agglutination  Reaction. — The  nearest  approach  to  a 
trustworthy  means  of  identification  is  the  specific  reaction  of 
typhoid  bacilli  with  the  blood  of  typhoid  subjects.  When 
typhoid  bacilli  are  brought  in  contact  with  the  blood  serum 


516     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

from  human  beings  sick  of  typhoid  fever,  or  from  animals 
that  have  survived  inoculation  with  cultures  of  this  organ- 
ism, there  occurs  a  peculiar  alteration  in  the  relation  of  the 
organisms  to  one  another  in  the  fluid.  As  ordinarily  seen 
in  a  hanging  drop  of  bouillon,  the  typhoid  bacilli  appear  as 
single,  actively  motile  cells;  when  to  such  a  drop  a  little 
dilute  serum  from  a  case  of  typhoid  fever  is  added  the  motility 
of  the  bacteria  gradually  lessens  and  finally  ceases,  and  they 
then  congregate,  "agglutinate"  in  larger  and  smaller  clumps, 
or  if  one  add  to  4  or  5  c.c.  of  a  twenty-four-hour-old  bouillon 
culture  of  typhoid  bacilli  in  a  narrow  test-tube  about  eight 
drops  of  serum  from  a  case  of  typhoid  fever  and  maintain 
this  mixture  at  body  temperature  the  normally  clouded 
culture  will  be  seen  after  a  few  hours  to  have  undergone  a 
change;  instead  of  a  diffuse  clouding  it  is  clear  and  floccu- 
lent  masses  of  the  bacteria  that  have  agglutinated  together 
as  a  result  of  the  specific  action  of  the  serum  used  will  be 
scattered  about  in  it. 

For  the  hanging-drop  test,  sufficient  serum  may  be 
obtained  from  a  needle-prick  in  the  finger,  while  for  the 
test-tube  reaction  a  larger  amount  is  needed;  this  may  be 
obtained  from  blood  drawn  from  a  superficial  vein  by  means 
of  a  hypodermic  syringe,  or  from  the  cleansed  skin  by  a  wet- 
cup,  or,  better  still,  from  a  small  cantharides  or  ammonia 
blister. 

It  is  proper  to  state,  however,  that  occasionally  cultures 
of  genuine  typhoid  bacilli  are  encountered  that  do  not 
respond  to  this  peculiar  influence  of  typhoid  blood,  even 
though  the  blood  be  tested  at  different  stages  of  the  disease, 
and  even  though  it  may  cause  the  characteristic  cessation 
of  motion  and  clumping  with  other  cultures  of  this  organism 
upon  which  it  is  tried. 


BACILLUS  TYPHOSUS  517 

"Widal's  Reaction." — When  employed  conversely — i.  e., 
for  deciding  if  the  serum  used  is  from  a  case  of  typhoid  fever 
or  not — the  reaction  constitutes  "  Widal's  serum  diagnosis 
of  typhoid  fever."  In  beginning  these  tests  it  is  often  neces- 
sary to  try  several  cultures  of  genuine  typhoid  bacilli  from 
different  sources  and  of  varying  degrees  of  vitality,  before 
a  strain  is  procured  that  reacts  conspicuously  and  quickly 
with  genuine  typhoid  serum. 

WIDAL'S  REACTION  WITH  DRIED  BLOOD. — This  reaction 
can  also  be  obtained  with  redissolved  dried  blood — i.  e., 
by  the  Johnston  method:  a  drop  of  the  blood  to  be  tested, 
obtained  by  a  needle-prick  in  the  cleansed  ringer  or  lobe  of 
the  ear,  is  collected  on  a  bit  of  clean,  unglazed  paper  and 
allowed  to  dry.  The  paper  is  then  folded,  kept  free  from 
contamination,  and  taken  to  the  laboratory.  With  a 
medium-size  platinum-wire  loop  a  drop  of  sterile  bouillon, 
water,  or  physiological  salt  solution  is  gently  rubbed  upon 
the  loop  of  dried  blood  until  the  contents  of  the  loop  are  of 
a  dark  amber  color;  this  is  then  mixed  with  a  drop  of 
bouillon  culture  of  typhoid  bacilli  on  a  cover-glass,  which  is 
mounted  upon  the  hollow-ground  slide  as  a  hanging  drop, 
when  the  effect  of  the  diluted  blood  upon  the  culture  can 
be  observed  with  the  microscope.  The  reaction,  if  positive, 
should  occur  within  a  half  hour.  Many  object  to  this 
method  because  it  is  impossible  accurately  to  dilute  the 
blood  by  the  plan  used.  A  number  of  tests  have  shown  us 
that  preparations  made  in  this  way  correspond  roughly 
with  a  fresh-blood  dilution  of  from  1  :  15  to  1  :  20,  as  deter- 
mined by  the  hemoglobinometer.  In  a  small  number  of 
cases  in  which  parallel  tests  were  made  with  this  and  with 
fresh  fluid  serum  the  results  were  concordant.  We  are 
inclined  to  the  opinion,  however,  that  in  doubtful  cases,  in 


518     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

which  all  the  available  clinical  evidence  is  opposed  to  either 
the  positive  or  negative  results  of  the  test,  the  difficulty  is 
much  more  certainly  cleared  away  by  the  use  of  highly  dilute 
and  exactly  diluted  fresh  serum  than  by  this  method.  Com- 
petent observers  are  of  the  opinion  that  in  all  such  cases 
the  quantity  of  serum  in  the  hanging  drop  should  be 
decreased  until  it  is  present  in  the  proportion  of  from  1  :  50 
to  1  :  60,  and  that,  if  after  exposure  to  this  dilution  for  two 
hours  the  bacilli  are  still  motile  and  not  clumped  together 
or  the  reaction  is  deficient  in  only  one  or  the  other  of  these 
peculiarities,  the  case  from  which  the  serum  was  obtained 
may  be  safely  regarded  as  not  typhoid  fever,  or  if  typhoid 
the  examination  was  not  made  at  a  time  when  agglutinin 
was  present  in  demonstrable  quantities  in  the  circulating 
blood. 

Experience  with  both  the  dry-blood  and  the  fresh  serum 
methods  show  the  culture  used  to  be  one  of  the  most  impor- 
tant factors  in  the  test.  After  deciding  upon  the  most 
suitable  culture  for  the  reaction — and  it  is  often  necessary 
to  try  a  great  number  from  various  sources — it  should  be 
transplanted  daily  into  fresh  bouillon  and  kept  at  a  tem- 
perature rarely  above  20°  or  22°  C.  The  bacilli  grown 
under  these  circumstances  are  usually  somewhat  longer 
than  when  cultivated  at  higher .  temperature,  and  they 
exhibit  a  regular,  gliding  motility  that  renders  it  more  easy 
to  follow  the  individual  cells  under  the  microscope  than 
when  they  possess  the  usual  active,  darting  motion. 

In  a  group  of  cases  examined  by  us  by  the  dry-blood 
method,  including  typhoid  and  other  febrile  conditions 
there  was  a  discrepancy  between  the  clinical  and  the  labora- 
tory diagnosis  in  from  2  to  3  per  cent,  of  the  cases  examined. 

In  the  hands  of  all  who  have  carefully  employed  the 


BACILLUS  TYPHOSUS  519 

Widal  reaction  for  the  diagnosis  of  typhoid  fever  the  results 
are  reported  to  have  been  almost  uniformly  satisfactory.  In 
the  great  majority  of  cases  the  reaction  is,  so  far  as  experi- 
ence indicates,  specific — i.  e.,  a  typical  reaction  does  not 
occur  between  typhoid  serum  or  blood  and  organisms  other 
than  the  typhoid  bacillus,  nor  between  the  typhoid  bacillus 
and  serums  other  than  those  from  cases  of  typhoid  fever. 
There  are,  however,  confusing  reactions — so-called  pseudo- 
reactions — in  which  more  or  less  clumping  of  the  bacilli 
and  a  diminution  of  motion,  without  complete  cessation, 
are  observed.  These  reactions  have  been  seen  to  occur 
with  normal  blood  and  with  blood  from  other  febrile  con- 
ditions. It  is  said  by  Johnston  and  McTaggart1  that  they 
can  be  prevented  if  cultures  of  just  the  proper  degree  of  vitality 
are  employed;  and  this  corresponds  with  the  results  of  a 
fairly  wide  personal  experience  with  the  test. 

In  the  light  of  present  experience  it  is  fair  presumptive 
evidence  that  the  serum  is  from  a  case  of  typhoid  fever 
when  unmistakable  agglutination  and  cessation  of  motion 
are  seen  in  from  fifteen  to  twenty  minutes  after  typhoid 
bacilli  are  mixed  with  the  serum  of  a  conspicuous  febrile 
condition. 

The  blood  of  certain  animals,  as  well  as  a  number  of 
chemical  substances,  such  as  corrosive  sublimate,  alcohol, 
salicylic  acid,  resorcin,  and  safranin  in  high  dilution,  cause 
agglutination  of  the  typhoid  bacilli;  but  the  reaction  is 
not  specific,  for  in  most  cases  they  have  the  same  effect  on 
other  motile  bacilli. 

Drinking  Water. — All  the  points  with  regard  to  morpho- 
logic and  biologic  characters  of  bacillus  typhosus,  and  of 
the  organisms  closely  resembling  it,  should  be  borne  in 

1  Montreal  Medical  Journal,  March,  1897. 


520     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

mind  in  the  examination  of  drinking-water  supposed  to  be 
contaminated  by  typhoid  dejections,  for  the  organisms  which 
most  closely  approach  the  typhoid  bacillus  in  growth  and 
morphology  are  just  those  organisms  which  would  appear 
in  water  contaminated  from  cesspools — i.  e.,  the  organisms 
constantly  found  in  the  normal  intestinal  tract.  Even  in 
the  stools  of  typhoid-fever  patients  the  presence  of  these 
normal  inhabitants  of  the  intestinal  tract  renders  the  isola- 
tion of  the  typhoid  organisms  somewhat  troublesome. 

Methods  of  Isolating  the  Typhoid  Bacillus. — From  the  fore- 
going it  is  obvious  that  bacillus  typhosus  is  so  variable  in 
many  of  its  biological  peculiarities,  and  is  so  closely  simu- 
lated in  certain  respects  by  a  group  of  other  organisms  to 
which  it  appears  to  be  botanically  related,  that  its  identi- 
fication, especially  outside  the  infected  body,  is  a  matter  of 
considerable  difficulty  and  uncertainty.  For  these  reasons 
many  efforts  have  been  made  to  discover  specific  cultural 
reactions  for  the  organism,  and  with  this  end  in  view  many 
methods  have  been  devised  for  its  isolation  from  water, 
feces,  sewage,  and  other  matters  believed  to  contain  it. 
None  of  them,  however,  have  given  general  satisfaction, 
and  many  have  proved  wholly  untrustworthy. 

In  deciding  upon  a  suitable  routine  these  are  several  points 
that  should  be  borne  in  mind : 

(a)  As  bacillus  typhosus  when  present  in  water,  feces, 
soil,  milk,  etc.,  is  always  numerically  in  the  minority,  as 
compared  with  other  organisms,  it  is  desirable  to  employ 
a  method  that  will  encourage  its  multiplication  without 
at  the  same  time  favoring  the  same  rate  of  multiplication 
by  other  organisms  present,  that  is  to  say,  to  use  an  "  enrich- 
ing medium;"  (6)  and  to  possess  a  method  that  will  make 
comparatively  simple  the  isolation  or  separation  of  the 


BACILLUS  TYPHOSUS  521 

typhoid  bacilli,  after  "enrichment,"  from  the  other  organ- 
isms with  which  it  is  associated.  With  these  objects  in 
mind  a  routine  that  gives  very  general  satisfaction  is  as 
follows : 

Enriching  Media. — For  this  purpose  ox  bile  and  "  brilliant 
green"  have  been  found  to  favor  the  growth  of  typhoid 
bacilli,  and  to  be  less  favorable  to  the  growth  of  other 
organisms  associated  with  it;  consequently  if  a  bit  of 
typhoid  feces  or  a  portion  of  infected  water  or  milk  be 
mixed  with  either  of  these  media  and  kept  at  suitable  tem- 
perature for  a  time,  the  result  will  be  a  more  conspicuous 
growth  of  bacillus  typhosus  than  of  the  other  organisms. 

Two  forms  of  ox  bile  may  be  employed: 

(1)  Pure  fresh  bile  direct  from  the  gall-bladder  of  a  freshly- 
slaughtered  ox,  or  (2)  a  solution  of  peptone  and  dried  ox 
bile  of  the  following  proportions: 

Dried  ox  bile       .      .      .      .      .      .      .      .      .      .      .10  parts 

Peptone 1  part 

Water 100  parts 

In  either  event  convenient  amounts  are  placed  in  test- 
tubes  and  sterilized;  after  which  they  are  ready  for  inocu- 
lation with  the  mixture  suspected  of  containing  the  typhoid 
bacillus.  After  inoculation  they  are  kept  at  body  tem- 
perature for  about  twenty-four  hours,  when  plates  may  be 
made  with  the  differential  media  to  be  described  below. 

Instead  of  the  ox  bile  the  aniline  dye  known  as  "brilliant 
green"  may  be  employed.  This  substance  suppresses  to 
some  extent  the  growth  of  organisms  other  than  bacillus 
typhosus,  particularly  those  of  the  colon  group.  It  is  used 
in  the  following  manner:  To  test-tubes  containing  a  known 
amount  (8  to  10  c.c.)  of  peptone  solution,  "brilliant  green"  is 


522     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

added  in  varying  amounts  so  as  to  have  a  series  of  solutions 
ranging  in  strength  from  one  part  of  the  green  to  500,000,  to 
one  part  to  100,000  of  the  peptone  solution.  A  convenient 
stock  solution  of  the  "brilliant  green"  is  1  : 1000  in  water. 
From  this  such  amounts  are  added  to  the  tubes  of  peptone 
solution  as  will  give  the  desired  series  of  dilutions.  The 
tubes  of  peptone  solution  should  have  been  sterilized  before 
the  green  is  added.  When  ready,  one  adds  to  each  of  these 
tubes  an  amount  of  the  substance  under  consideration: 
if  it  be  feces — a  moderate  loopful  may  be  broken  up  in  1  c.c. 
of  bouillon  and  one  or  two  loopfuls  of  this  used;  if  it  be  water 
or  milk  from  0.1  to  0.3  c.c.  The  amount  best  suited  must 
be  determined  by  experiment. 

When  inoculated  the  tubes  are  kept  at  body  temperature 
for  from  eighteen  to  twenty-four  hours,  when  they  are 
ready  for  the  "differential"  or  "selective"  plating. 

The  enriching  media  should  be  free  of  sugar. 

In  the  process  of  plating,  specially  prepared  selective 
media  are  used  that  aim  to  render  evident  to  the  naked  eye 
distinguishing  differences  between  the  colonies  of  bacillus 
typhosus  and  those  of  other  confusing  organisms,  Of  a 
number  of  special  media  employed  for  this  purpose  two  have 
proved  very  satisfactory — notably  that  recommended  by 
Drigalski  and  Conradi,  and  that  by  Endo. 

METHOD  OF  v.  DRIGALSKI  AND  CoNRADi.1 — This  method 
aims  to  separate  bacillus  typhosus  from  bacillus  coli  on  the 
basis  of  their  fermenting  properties,  in  such  a  manner  as  not 
to  hinder  the  growth  of  bacillus  typhosus,  but  rather  to 
make  the  conditions  for  its  growth  as  favorable  as  possible. 

The  authors  give  the  following  directions  for  the  prepara- 
tion of  their  culture  medium : 

1  Zeitschrift  fur  Hygiene,  1902,  Bd.  xxxix,  p.  288. 


BACILLUS  TYPHOSUS  523 

a.  Preparation  of  agar:  1500  grams  of  finely  chopped  beef 
are  placed  in  two  liters  of  water  and  set  aside  for  twenty- 
four  hours.    This  meat  infusion  is  then  boiled  for  one  hour, 
filtered,  and    20  grams  of    Witte's  peptone,  20  grams  of 
nutrose,  and  10  grams  of  sodium  chloride  are  added  and 
again  boiled  for  an  hour,  filtered,  and  60  grams  of  agar-agar 
are  added,  boiled  for  three  hours  (or  one  hour  in  the  auto- 
clave), rendered  slightly  alkaline  to  litmus  paper,  filtered, 
and  boiled  for  one-half  hour. 

b.  Litmus  solution:  (Litmus  solution  according  to  Kubel 
and   Tiemann)   260  c.c.,  boil  ten  minutes,  add  30   grams 
chemically  pure  lactose,  boil  fifteen  minutes. 

c.  The  hot  litmus-lactose  solution  is  added  to  the  hot 
nutritive  agar,  thoroughly  mixed,  and  the  alkaline  reaction 
is  again  restored.    To  this  medium  is  then  added  4  c.c.  of 
a  hot  sterile  solution  of  10  per  cent,   water-free  sodium 
carbonate,  20  c.c.  of  freshly  prepared  solution  of  0.1  gram 
crystal  violet  (Hochst)  in  100  c.c.  of  warm  sterile  distilled 
water. 

One  now  has  a  meat-infusion-peptone-nutrose-agar  with 
13  per  cent,  of  litmus  solution  and  0.01  per  thousand  crys- 
tal violet.  It  becomes  very  hard  on  solidifying,  without 
becoming  too  dry.  Plates  are  poured  of  this  material  and 
held  in  readiness  for  some  time,  and  the  remainder  of  the 
medium  is  preserved  in  flasks  in  portions  of  200  c.c.  each. 

If  the  lactose  is  boiled  for  a  longer  time  than  directed 
it  is  reduced,  with  an  acid  reaction  of  the  culture  medium, 
and  the  content  in  lactose  falls  below  the  required  quantity, 
and  the  alteration  in  the  color  of  the  colon  colonies  appears 
too  early.  For  this  reason  it  is  also  necessary  to  liquefy 
the  agar  as  quickly  as  possible  in  pouring  plates  from  the 
agar  medium  stored  in  flasks. 


524     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

In  employing  this  culture  medium  it  is  necessary  to  have 
a  uniform  suspension  of  a  portion  of  the  material  to  be 
examined  and  to  make  a  series  of  plate  inoculations  from 
this  suspension  by  smearing  carefully  the  material  under 
consideration  over  the  surface  of  the  medium  in  the  plates,  a 
sterile  platinum  spatula  or  a  sterile  bent  glass  rod  being  used 
for  the  purpose. 

After  fourteen  to  sixteen  hours  at  37°  C.,  and  still  better 
after  twenty  to  twenty-four  hours,  the  cultures  are  readily 
differentiated : 

a.  Bacillus  Coli:  All  cultures  of  true  colon  that  have  been 
examined  form  colonies  of  2  to  6  or  more  millimeters  in 
diameter,  of  reddish  color  and  translucent.     In  each  intes- 
tinal   evacuation    there    are    usually    several    varieties    of 
colon  colonies  which  differ  according  to  their  size  and  tex- 
ture, translucency,  and-  the  intensity  of  the  alteration  of 
the  color  which  they  bring  about.     Many  colon  colonies 
are  bright  red,  some  are  cloudy,  and  others  are  quite  opaque, 
dark-wine  red  in  color,  while  still  others  form  large  colonies 
which  are  surrounded  by  a  red  halo. 

b.  Bacillus  Typhosus:  The  colonies  have  a  diameter  of 
1  to  3  millimeters,  rarely  larger.    Their  color  is  blue,  with 
a  tendency  toward  violet.    In  structure  they  are  glistening, 
with  a  single  contour,  somewhat  of  the  nature  of  a  dew  drop. 
Only  in  isolated  instances  is  the  colony  larger  and  more 
cloudy  in  appearance. 

The  Endo  Media. — (Modification  of  Kendall  and  Day.) 
Prepare  the  following : 

(a)  Water 1000  c.c. 

Powdered  agar-agar 15  grams 

Peptone  (Witte) 10  grams 

Meat  Extract  (Liebig) 3  grams 


BACILLUS  TYPHOSUS  525 

Heat  until  the  agar-agar  is  dissolved,  keeping  the  mass  to 
100  c.c.  volume  by  addition  of  water.  This  should  require 
about  an  hour  over  the  flame,  or  less  if  the  mass  be  dissolved 
in  the  autoclave.  Render  just  alkaline  to  litmus  by  the 
addition  of  decinormal  sodium  hydroxide  solution.  Filter 
and  decant  to  flasks  containing  100  c.c.  each.  Sterilize. 

(b)  Prepare  a  10  per  cent,  solution  of  fuchsin  in  96  per 
cent,  alcohol. 

(c)  Prepare  a  10  per  cent,  solution  of  sodium  sulphite  in 
water. 

For  the  making  of  the  plates  mix  1  c.c.  of  (b)  with  10  c.c. 
of  (c)  and  heat  in  the  steam  sterilizer  (100°  C.)  for  20  minutes. 
This  decolorizes  the  fuchsin.  To  each  100  c.c.  of  the  agar 
prepared  as  (a),  add  1  per  cent,  of  chemically  pure  lactose 
and  heat  in  the  steam  sterilizer  at  100°  C.  until  the  agar- 
agar  is  completely  liquefied  and  the  lactose  dissolved.  To 
each  100  c.c.  of  this  lactose-agar  add  1  c.c.  of  the  decolorized 
fuchsin  solution,  mix  thoroughly  and  while  still  fluid  and 
warm,  pour  into  sterile  Petri  dishes;  sufficient  in  each  dish 
to  give  a  layer  of  from  3  to  5  mm.  depth.  Place  these  dishes, 
with  the  covers  removed,  in  the  incubator  until  the  agar- 
agar  has  set;  this  will  require  about  30  minutes.  They  are 
then  ready  for  inoculation.  The  plates  are  now  inoculated 
by  spreading  evenly  over  the  surface  small  quantities  from 
the  primary  "enriching"  cultures.  This  is  best  done  by 
the  use  of  a  bent  glass  rod  that  has  been  sterilized  in  the 
flame  and  allowed  to  cool. 

If  typhoid  bacilli  be  present  they  develop  as  tiny,  trans- 
parent, practically  colorless  colonies  of  from  1  to  2  mm. 
in  diameter.  Colonies  of  the  colon  or  paracolon  group 
appear  as  larger,  denser  pink  or  red  massses  and  cause 
a  reddening  of  the  medium  about  them. 


526     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

All  small,  transparent,  colorless  colonies,  i.  e.,  those,  sug- 
gestive of  bacillus  typhosus  are  to  be  isolated  in  pure  cul- 
ture and  identified  by  the  usual  procedures. 

Precipitation  Method  of  Ficker.1 — Two  liters  of  the  water 
to  be  examined  are  placed  in  a  narrow  sterile  glass  cylinder 
and  rendered  alkaline  with  8  c.c.  of  10  per  cent,  sodium 
carbonate  solution,  and  afterward  7  c.c.  of  a  10  per  cent, 
sulphate  of  iron  solution  are  added  and  mixed  with  the 
water  by  means  of  a  sterile  glass  rod.  The  cylinder  is  then 
placed  in  the  ice-chest.  Precipitation  is  complete  in  two 
to  three  hours.  The  overstanding  water  is  syphoned  off, 
and  the  precipitate  or  portions  thereof  are  poured  into 
sterile  test-tubes.  To  this  precipitate  is  now  added  about 
a  half  volume  of  a  25  per  cent,  solution  of  neutral  potassium 
tartrate.  The  test-tube  is  closed  with  a  sterile  rubber  cork 
and  the  mixture  thoroughly  agitated,  whereby  the  precipi- 
tate is  completely  dissolved.  With  a  sterile  pipette  one  part 
of  this  fluid  is  mixed  in  a  test-tube  with  two  parts  of  sterile 
bouillon,  and  this  mixture  is  distributed  over  a  series  of 
Drigalski-Conradi  plates.  Ficker  advises  when  possible 
the  use  of  a  centrifuge  for  the  separation  of  the  precipitate, 
as  he  believes  the  results  are  likely  to  be  more  satisfactory. 

Prophylactic  Vaccination. — That  typhoid  fever  may  be 
prevented  by  vaccination  is  an  accomplished  fact.  Expe- 
rience gathered  during  the  past  few  years  by  all  civilized 
governments,  notably  those  of  England,  France,  Germany 
and  this  country  is  unanimous  in  support  of  this  statement. 

No  argument  could  be  more  convincing  than  the  results 
obtained  through  the  vaccinations  practiced  in  the  United 
States  Army  and  Navy,  where  the  procedure  is  com- 
pulsory. The  following  abstract  from  one  of  the  several 

1  Hygienische  Rundschau,  1904,  Bd.  xiv,  S.  7. 


BACILLUS  TYPHOSUS  527 

excellent  reports  submitted  by  Major  Russell  of  the  U.  S. 
Army  Medical  Corps,  suffices  to  illustrate  the  protective 
value  of  antityphoid  vaccination: 

In  1898,  during  the  Spanish- American  War,  when  no 
preventive  vaccination  was  practised,  there  were  assembled 
at  Jacksonville,  Florida,  10,759  troops,  among  whom  there 
were  certainly  1729  cases  of  typhoid  fever,  and  including 
those  cases  that  were  probably  typhoid  fever,  this  figure 
is  increased  to  2,693  cases  with  248  deaths.  Contrast  that 
with  the  following: 

In  1911  there  were  assembled  for  maneuvers  along  the 
Mexican  frontier  about  20,000  United  States  troops.  All 
were  vacinnated  against  typhoid  fever;  with  the  result 
that  after  four  months  in  camps  (about  the  same  time  as 
the  men  remained  in  the  Jacksonville  camp)  there  developed 
one  case  of  typhoid  fever.  This  case  did  not  prove  fatal. 
It  should  be  said  that  the  disease  was  known  to  exist  among 
residents  in  the  immediate  vicinity  of  this  camp  and  that 
the  soldiers  were  allowed  free  access  to  the  infected 
districts. 

By  the  adoption  of  compulsory  vaccination  in  the  Army, 
typhoid  fever  has  been  practically  eliminated.  For  the 
entire  United  States  the  typhoid  mortality  for  the  year  1913 
was  at  the  rate  of  12.7  per  100,000,  while  for  the  entire 
army  it  was  0  per  100,000.* 

It  is  needless  to  pursue  the  argument  further;  though  it 
should  be  said  that  the  vaccination  is  harmless  to  the 
individual. 

Major  Whitmore  of  the  Medical  Corps  of  the  United 
States  Army  states  that  of  130,000  adults  vaccinated,  97 
per  cent,  gave  no  disagreeable  reaction. 

1  For  a  discussion  of  typhoid  fever  during  the  war  see  Annual  Reports  of 
the  Surgeon-General  of  the  Army,  1919  and  1920. 


528     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

Major  Russell  has  also  shown  by  a  very  careful  study  that 
children  under  five  years  of  age  may  be  safely  vaccinated 
if  appropriate  doses  of  the  vaccine  be  employed. 

The  Vaccine. — The  agent  used  in  vaccination  is  typhoid 
bacilli  that  have  been  killed  by  heat.  In  some  instances 
living,  sensitized  typhoid  bacilli  have  been  employed  with 
good  results,  but  as  the  bulk  of  experience  has  been  obtained 
with  the  dead  cultures  and  as  this  is  much  the  more  simple 
procedure  it  is  probable  that  it  is  the  method  that  will  be 
generally  adopted. 

The  vaccine  is  prepared  as  follows:  A  proven  culture  of 
bacillus  typhosus  is  grown  on  nutrient  agar-agar  at  body 
temperature  for  eighteen  to  twenty  hours.  The  growth  is 
then  carefully  washed  from  the  surface  with  a  small  quantity 
of  sterile  physiological  salt  solution.  This  emulsion  is  then 
heated  in  a  water  bath  to  53°  C.  for  one  hour,  after  which 
it  is  diluted  with  sterile  salt  solution  to  a  point  at  which  a 
billion  bacilli  are  contained  in  a  cubic  centimeter  of  the 
emulsion.  Finally  tricresol  in  the  proportion  of  0.25  per 
cent,  is  added  as  a  preservative.  Before  using  such 
vaccine  its  safety:  i.  e.,  its  freedom  from  objectionable 
qualities,  especially  from  the  germs  of  tetanus,  is  invariably 
tested,  as  is  also  its  efficiency  in  calling  forth  the  customary 
reactions  of  intoxication  and  resistance.  These  tests  are  made 
upon  such  sensitive  reagents  as  mice,  guinea-pigs,  and  rabbits. 

The  original  vaccination  consisted  in  the  subcutaneous 
injection  of  a  volume  of  emulsion  equivalent  to  500  million 
bacilli  followed  on  the  tenth  and  twentieth  days  with  doses 
equivalent  to  1000  million  bacilli;  that  is  to  say,  the  first 
dose  is  0.5  c.c.  of  the  above-mentioned  emulsion,  while 
the  second  and  third  doses  are  1  c.c.  each. 


BACILLUS  TYPHOSUS  529 

As  a  rule  the  injections — particularly  the  primary  one — 
are  followed  by  a  red,  tender,  swollen  area  at  the  site  of 
puncture.  This  may  be  accompanied  by  headache,  fever, 
general  malaise  and  sometimes  by  a  chill  with  vomiting 
or  diarrhea.  In  the  majority  of  individuals  the  reactions 
are  mild  and  disappear  in  from  thirty-six  to  forty-eight 
hours. 

In  the  later  use  of  this  vaccine  it  was  found  possible  to 
secure  the  desired  protection  by  one  single  injection,  instead 
of  three.  In  from  one  to  three  persons  out  of  every  thousand 
vaccinated  the  reaction  may  be  severe,  though  they  are 
not  dangerous.  No  ill  effects  of  a  permanent  nature  have 
thus  far  been  noted  in  the  thousands  of  civilians  and  millions 
of  soldiers  inoculated  during  the  war,  nor  have  the  vaccina- 
tions been  seen  to  influence  unfavorably  the  course  of  other 
diseases  from  which  the  individual  may  be  suffering. 

It  should  be  needless  to  say  that  strict  aseptic  precau- 
tions are  to  be  taken  in  performing  the  operation.  The 
resistance  that  is  excited  by  the  vaccination  is  an  "active 
immunity" — that  is,  it  is  an  immunity  identical  in  nature 
with  that  acquired  by  an  individual  who  has  recovered  from 
an  attack  of  typhoid  fever.  In  so  far  as  can  be  stated  now, 
however,  the  immunity  is  not  permanent.  All  indications 
point  to  its  gradual  diminution  and  possible  disappearance 
often  in  two  to  three  years,  so  that  revaccination  after  the 
lapse  of  this  time  is  advisable. 

NOTE. — Obtain  a  pure  culture  of  typhoid  bacilli,   and 
from  this  make  inoculations  upon  a  series  of  potatoes  of 
different  ages   and   from   different   sources.     Do  they  all 
grow  alike  ? 
34 


530     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

Before  sterilizing  render  another  lot  of  potatoes  slightly 
acid  with  a  few  drops  of  very  dilute  acetic  acid;  render 
others  very  slightly  alkaline  with  dilute  caustic  soda.  Are 
any  differences  in  the  growths  noticeable  ? 

Make  a  series  of  twelve  tubes  of  peptone  solution  to  which 
rosolic  acid  has  been  added.  Inoculate  them  all  with  as 
nearly  the  same  amount  of  material  as  possible  (one  loopful 
from  a  bouillon  culture  into  each  tube) ;  place  them  all  in  the 
incubator.  Is  the  color-change,  as  compared  with  that  of 
the  control-tube,  the  same  in  all  cases  ? 

Compare  the  morphology  of  cultures  of  the  same  age  on 
gelatin,  agar-agar,  and  potato. 

Select  a  culture  in  which  the  vacuolations  are  quite  marked. 
Examine  this  culture  unstained.  Do  the  organisms  look 
as  if  they  contained  spores?  How  would  you  demonstrate 
that  the  vacuolations  are  not  spores?  What  is  the  crucial 
test  for  spores? 

Obtain  from  normal  feces  a  pure  culture  of  the  com- 
monest organism  present.  Write  a  full  description  of  it. 
Now  make  parallel  cultures  of  this  organism  and  of  the 
typhoid  bacillus  on  all  the  different  media?  How  do  they 
differ?  In  what  respects  are  they  similar? 

BACILLUS   COLI    (ESCHERICH),   MIGULA,    1900. 

SYNONYMS:  Bacillus  neapolitanus,  Emmerich,  1884;  Bacillus  pyogenes 
fcetidus,  Passet,  1885;  Emmerich's  bacillus,  Eisenberg,  1886;  Bacterium 
coli  commune,  Escherich,  1886. 

This  organism  was  discovered  by  Escherich,  in  1886,  in 
the  intestinal  discharges  of  milk-fed  infants.  It  has  since 
been  demonstrated  to  be  a  constant  inhabitant  of  the  intes- 
tines of  man  and  domestic  animals,  and  is,  therefore,  con- 
sidered a  commensal  species. 


BACILLUS  COLI  531 

For  a  time  after  its  discovery  it  was  considered  of  but 
little  importance  and  attracted  attention  only  because  of 
its  resemblance,  in  certain  respects,  to  the  bacillus  of  typhoid 
fever,  with  which  it  was  occasionally  confounded.  In  this 
particular  it  still  serves  as  a  subject  for  study.  Some  have 
even  gone  so  far  as  to  regard  them  as  but  varieties  of  one  and 
the  same  species,  though  in  the  present  state  of  our  knowl- 
edge this  is*  an  assumption  for  which  as  yet  there  are  not 
sufficient  grounds.  That  they  possess  in  common  certain 
general  points  of  resemblance  and  often  approach  one 
another  in  some  of  their  biological  peculiarities  is  true;  but, 
as  we  shall  learn,  they  each  possess  peculiarities  which, 
when  considered  together,  render  their  differentiation  from 
one  another  a  matter  of  but  little  difficulty. 

With  the  wider  application  of  bacteriological  methods 
to  the  study  of  pathological  processes  it  was  occasionally 
observed  that,  under  favorable  circumstances,  bacillits  coli 
disseminated  from  its  normal  habitat  and  appeared  in 
remote  organs,  often  associated  with  diseased  conditions. 
This  was  at  first  considered  of  but  little  importance,  and  its 
presence  in  these  localities  was  viewed  as  accidental.  Its 
repeated  appearance,  however,  in  different  organs  of  the 
body  and  the  frequency  of  its  association  with  pathological 
conditions,  ultimately  attracted  attention  to  it,  and  in 
consequence  a  great  deal  has  been  written  concerning  the 
possible  pathogenic  nature  of  this  organism. 

The  fact  that  it  is  a  commensal  species,  always  intimately 
associated  with  certain  of  our  life-processes,  together  with 
the  fact  that  it  is  known  to  appear  in  organs  other  than 
that  in  which  it  is  normally  located,  and  that  its  occurrence 
in  diseased  conditions  is  not  rare,  justifies  the  opinion  that 
it  is  one  of  the  most  important  of  the  microorganisms  with 
which  we  have  to  deal. 


532      APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

While  not  generally  considered  a  pathogenic  organism, 
there  is,  nevertheless,  sufficient  evidence  to  warrant  the 
statement  that  under  favorable  conditions  of  reduced  vitality 
on  the  part  of  the  animal  tissues,  this  organism  may  assume 
pathogenic  properties,  so  that  its  presence  in  diseased  con- 
ditions is  not  always  to  be  considered  as  accidental,  though 
this  is  frequently  the  case. 

The  morphological  and  cultural  peculiarities  of  bacillus 
coli  are  as  follows : 

Morphology. — In  shape  it  is  a  rod  with  rounded  ends, 
sometimes  so  short  as  to  appear  almost  spherical,  while 
again  it  is  seen  as  very  much  longer  threads.  Often  both 
forms  are  associated  in  the  same  culture.  It  may  occur  as 
single  cells,  or  as  pairs  joined  end  to  end. 

It  has  no  peculiar  morphological  features  that  can  aid 
in  its  identification.  It  is  usually  said  to  be  motile,  and 
undoubtedly  is  motile  in  the  majority  of  cases;  but  its 
movements  are  at  times  so  sluggish  that  a  positive  opinion 
is  often  difficult. 

By  Loffler's  method  of  staining,  flagella  can  be  demon- 
strated, though  usually  not  in  such  numbers  as  are  seen  to 
occur  on  the  typhoid  fever  bacillus. 

Cultural  Characteristics. — It  grows  both  with  and  without 
free  oxygen. 

On  the  surface  of  gelatin  its  colonies  appear  as  small,  dry, 
irregular,  flat,  blue-white  points  that  are  commonly  some- 
what dentated  or  notched  at  the  margin.  They  are  a  trifle 
denser  at  the  center  than  at  the  periphery,  and  are  often 
marked  at  or  near  the  middle  by  an  oval  or  round  nucleus- 
like  mass — the  original  colony  from  which  the  layer  on  the 
surface  developed.  When  located  in  the  depths  of  the 
gelatin,  and  examined  with  a  low-power  lens,  they  are  at 


BACILLUS  CO  LI  533 

first  seen  to  be  finely  granular  and  of  a  very  pale  greenish- 
yellow  color;  later  they  become  denser,  darker,  and  much 
more  markedly  granular;  in  shape  they  are  round,  oval, 
and  lozenge-like.  When  the  surface  colonies  are  viewed 
under  a  low  power  of  the  microscope  they  present  essen- 
tially the  same  appearance  as  that  given  for  the  colonies 
of  the  bacillus  of  typhoid  fever,  viz.,  they  resemble  flattened 
pellicles  of  glass-wool,  or  patches  of  finely  ground  colorless 
glass.  Colonies  of  this  organism  on  gelatin  are  frequently 
encountered  that  cannot  be  distinguished  from  those  result- 
ing from  the  growth  of  bacillm  typhosus;  although,  as  a  rule, 
their  growth  is  a  little  more  luxuriant. 

In  stab-  and  smear-cultures  on  gelatin  the  surface-  growth 
is  flat,  dry,  and  blue-white  or  pearl  color.  Limited  growth 
occurs  along  the  track  of  the  needle  in  the  depths  of  the 
gelatin.  As  the  culture  becomes  older  the  gelatin  round 
about  the  surface-growth  may  gradually  lose  its  trans- 
parency and  become  cloudy,  often  quite  opaque.  In  still 
older  cultures  small  root-  or  branch-like  projections  from 
the  surface-growth  into  the  gelatin  are  sometimes  seen. 
At  times  these  may  be  of  a  distinctly  crystalline  appear- 
ance. 

It  does  not  cause  liquefaction  of  gelatin. 

Its  growth  on  nutrient  agar-agar  and 'on  blood-serum  is 
luxuriant,  but  not  characteristic. 

In  bouillon  it  causes  diffuse  clouding  with  sedimentation. 
In  some  bouillon  cultures  an  attempt  at  pellicle  formation 
on  the  surface  may  be  seen,  but  this  is  exceptional.  In  old 
bouillon  cultures  the  reaction  becomes  alkaline  and  a  decided 
fecal  odor  may  be  detected. 

Its  growth  on  potato  is  rapid  and  voluminous,  appearing 
after  twenty-four  to  thirty-six  hours  in  the  incubator  as  a 


534     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

more  or  less  lobulated  layer  of  a  drab,  dark-cream,  or 
brownish-yellow  color. 

In  neutral  milk  containing  a  little  litmus  tincture  the 
blue  color  is  changed  to  red  after  from  eighteen  to  twenty- 
four  hours  in  the  incubator,  and,  in  addition,  the  majority 
of  cultures  cause  firm  coagulation  of  the  casein  in  about 
thirty-six  hours,  though  frequently  this  takes  longer.  Very 
rarely  the  litmus  may  indicate  the  production  of  acid  and  no 
coagulation  occur. 

In  media  containing  glucose  it  grows  rapidly  and  causes 
active  fermentation,  with  liberation  of  carbonic  acid  and 
hydrogen.  If  cultivated  in  solid  media  to  which  glucose 
(2  per  cent.)  has  been  added,  the  gas-formation  is  recognized 
by  the  appearance  of  numerous  bubbles  along  and  about 
the  points  of  growth.  If  cultivated  in  fluid  media,  also 
containing  glucose,  in  the  fermentation-tube,  evidence  of 
fermentation  is  given  by  the  collection  of  gas  in  the  closed 
arm  of  the  tube. 

On  lactose-litmus-agar-agar  its  colonies  are  pink  and  the 
color  of  the  surrounding  medium  is  changed  from  blue  to  red. 

In  Dunham's  peptone  solution  it  produces  indol  in  from 
forty-eight  to  seventy-two  hours. 

It  stains  with  the  ordinary  aniline  dyes.  It  is  decolorized 
when  treated  by  the  method  of  Gram. 

By  comparing  what  has  been  said  of  bacillus  typhosus 
and  of  bacillus  coll  it  will  be  seen  that,  while  they  simulate 
each  other  in  certain  respects,  they  nevertheless  possess 
individual  characteristics  by  which  they  may  readily  be 
differentiated.  The  least  variable  of  the  differential  points 
are: 

1.  Motility  of  bacillus  typhosus  is  much  more  conspicuous, 
as  a  rule,  than  is  that  of  bacillus  coli. 


BACILLUS  COLI  535 

2.  On  gelatin,  colonies  of  the  typhoid  bacillus  develop 
more  slowly  than  do  those  of  the  colon  bacillus. 

3.  On  potato,  the  growth  of  the  typhoid  bacillus  is  usually 
invisible    (though   not   always);  while   that   of   the   colon 
bacillus  is  rapid,  luxuriant,  and  always  visible. 

4.  The  typhoid  bacillus  does  not  cause  coagulation  of 
milk  with  acid  reaction.     The  colon  bacillus  does  this  in 
from  thirty-six  to  forty-eight  hours  in  the  incubator. 

5.  The  typhoid  bacillus  never  causes  fermentation,  with 
liberation  of  gas,  in  media  containing  glucose,  lactose,  or 
saccharose.    The  colon  bacillus  is  conspicuous  for  its  power 
of  causing  gaseous  fermentation  in  such  solutions. 

6.  In  nutrient  agar-agar  or  gelatin  containing  lactose  and 
litmus  tincture,  and  of  a  slightly  alkaline  reaction,  the  color 
of  the  colonies  of  typhoid  bacillus  is  pale  blue,  and  there  is 
no  reddening  of  the  surrounding  medium;  while  colonies  of 
the  colon  bacillus  are  pink  and  the  medium  round  about 
them  becomes  red. 

7.  The  typhoid  bacillus  does  not,  us  a  rule,  possess  the 
property  of  producing  indol  in  solutions  of  peptone;  the 
growth  of  the  colon  bacillus  in  these  solutions  is  accompanied 
by  the  production  of  indol  in  from  forty-eight  to  seventy- 
two  hours  at  37°  to  38°  C. 

Animal  Inoculations. — As  with  the  bacillus  of  typhoid 
fever,  the  results  of  inoculation  of  animals  with  cultures 
of  this  organism  cannot  be  safely  predicted.  According  to 
numerous  observers  the  effects  that  do  appear  are  in  most 
instances  to  be  attributed  to  the  toxic  rather  than  to  the 
infective  properties  of  the  culture  used. 

When  introduced  into  the  subcutaneous  tissues  of  mice 
it  has  no  effect,  while  similar  inoculations  of  guinea-pigs 
are  sometimes  (not  always)  followed  by  abscess  formation 


536      APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

at  the  point  of  operation,  or  by  alterations  very  similar  to 
those  produced  by  intra vascular  inoculation,  viz.,  death  in 
less  than  twenty-four  hours,  accompanied  by  redness  of 
the  peritoneum  and  marked  hyperemia  and  ecchymoses  of 
the  small  intestine,  together  with  swelling  of  Peyer's  patches. 
The  cecum  and  colon  may  remain  unchanged  or  present 
enlarged  follicles.  There  may  or  may  not  be  an  accumula- 
tion of  fluid  in  the  abdominal  cavity;  but  peritonitis  is 
rarely  present.  The  small  intestine  may  contain  bloody 
mucus. 

Intravenous  inoculation  of  rabbits  may  be  followed  by 
similar  changes,  with  often  the  occurrence  of  diarrhea 
before  death,  which  may,  in  the  acute  cases,  result  in  from 
three  to  forty  hours.  In  another  group  of  cases  acute  fatal 
intoxication  does  not  result,  and  the  animal  lives  for  weeks 
or  months,  dying  ultimately  of  what  appears  to  be  the 
effects  of  a  slow  or  chronic  form  of  infection.  For  a  few 
hours  after  inoculation  these  animals  present  no  marked 
symptoms;  exceptionally,  somnolence  and  diarrhea  have 
been  observed  at  this  period,  indicating  acute  intoxication 
from  which  the  animal  has  recovered.  The  affection  is 
unattended  by  fever.  The  most  marked  symptom  is  loss  of 
weight.  This  is  usually  progressive  from  'the  first  or  second 
day  after  inoculation,  with  slight  fluctuations  until  death. 

At  autopsy  the  animal  is  found  to  be  emaciated.  The 
subcutaneous  tissues  and  the  muscles  appear  pale  and  dry. 
The  serous  cavities,  particularly  the  pericardial,  may  con- 
tain an  excess  of  serum.  The  viscera  are  anemic.  The 
spleen  is  small,  thin,  and  pale.  Exceptionally  ulcers  and 
ecchymoses  are  observed  in  the  cecum,  but  generally  there 
are  no  lesions  of  the  intestinal  tract. 

The   most    striking   and    constant   lesions,    those    most 


BACILLUS  CO  LI  537 

characteristic  of  the  affection,  are  in  the  bile  and  in  the 
liver;  in  some  cases  the  quantity  of  bile  may  not  exceed 
the  normal,  but  in  others  the  gall-bladder  may  be  abnor- 
mally distended  with  bile.  The  bile  is  nearly  colorless  or 
has  a  pale  yellowish  or  brownish  tint,  with  little  or  no 
greenish  color.  Its  consistence  is  much  less  viscid  than 
normal,  being  often  thin  and  watery.  It  usually  contains 
small,  opaque,  yellowish  particles  or  clumps  which  can  be 
seen  floating  in  it,  even  through  the  walls  of  the  gall-bladder. 
These  clumps  consist  microscopically  of  bile-stained,  appar- 
ently necrotic,  epithelial  cells;  leukocytes  in  small  numbers; 
amorphous  masses  of  bile-pigment,  and  bacteria  often  in 
zooglea-like  clumps.  Similar  material  is  found  in  the  larger 
bile-ducts. 

The  liver  frequently  contains  opaque,  whitish  or  yellow- 
ish-white spots  and  streaks  of  irregular  size  and  shape,  which 
give  a  peculiar  mottling  to  the  organ  when  present  in  large 
number.  These  areas  may  be  numerous,  or  only  one  or  two 
may  be  found.  In  size  they  range  from  minute  points  to 
areas  of  from  2  to  3  cm.  in  extent.  By  microscopic  exami- 
nation they  are  found  to  represent  localities  where  the  liver- 
cells  have  undergone  necrosis  accompanied  by  emigration 
of  leukocytes,  and  the  cells  about  them  are  in  a  condition 
of  fatty  degeneration.  In  sections  of  the  liver  masses  of 
the  bacilli  may  be  discovered  in  and  about  the  necrotic 
foci  just  described. 

At  these  autopsies  the  colon  bacillus  is  not  found  generally 
distributed  through  the  body,  but  is  only  to  be  detected  in 
the  bile,  liver,  and  occasionally  in  the  spleen.1 

1  Consult  paper  by  Blachstein  on  this  subject,  Johns  Hopkins  Hospital 
Bulletin,  1891,  ii,  96. 


538     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

BACILLUS   PARATYPHOSUS. 

During  recent  years  careful  bacteriological  examination 
of  cases  of  continued  fever,  the  blood  from  which  had  no 
agglutinating  action  upon  typhoid  bacillus,  has  revealed  a 
group  of  bacilli  which  differ  from  bacillus  typhosus  in  certain 
important  particulars.  These  bacteria  possess  characters 
which  are  intermediate  between  those  of  bacillus  typhosus 
and  bacillus  coli,  some  resembling  more  closely  the  former, 
others  the  latter,  and  for  these  reasons  they  have  sometimes 
been  denominated  the  intermediate,  "near"  or  "para" 
group.  Some  of  the  organisms  isolated  from  such  cases 
of  continued  fever  resemble  very  closely  bacillus  enteriditis, 
which  Gaertner  found  in  cases  of  meat  poisoning. 

The  general  opinion  is  that  these  organisms  produce  a 
form  of  infection  sometimes  resembling  in'  many  of  its 
clinical  characters  that  produced  by  bacillus  typhosus. 
The  infection,  however,  is  usually  of  a  milder  type  and 
only  a  comparatively  small  number  of  cases  have  terminated 
fatally,  so  that  the  pathology  of  the  disease  is  not  well 
known.  Moreover,  the  biological  characters  of  the  different 
organisms  isolated  from  cases  of  paratyphoid  fever,  as  the 
condition  is  called,  show  such  wide  variations  that  it  is 
probable  the  pathology  of  different  cases  also  varies  with 
the  particular  type  of  organism  causing  the  infection. 

Buxton1  was  one  of  the  first  to  make  a  careful  compara- 
tive study  of  the  morphology  and  biology  of  this  group  of 
organisms.  He  classifies  the  intermediary  group  of  organ- 
isms in  the  following  manner: 

"  Paracolons :  those  which  do  not  cause  typhoidal  symp- 

1  Journal  of  Medical  Research,  viii,  201. 


BACILLUS  PARATYPHOSUS  539 

toms  in  man.  A  group  containing  numerous  different 
members,  but  culturally  alike. 

"  Paratyphoids :  those  which  cause  typhoidal  symptoms. 

"  (a)  A  distinct  species  culturally  unlike  the  paracolons. 

"(6)  A  distinct  species  culturally  resembling  the  para- 
colons." 

Buxton  and  others  state  that  some  of  those  producing 
typhoidal  symptoms  cannot  be  distinguished  culturally 
from  some  members  of  the  paracolon  group.  All  the  organ- 
isms of  this  intermediate  group  have*  the  morphological 
characters  of  the  colon-typhoid  group  of  organisms,  and 
they  cannot,  therefore,  be  distinguished  from  one  another 
by  the  form  or  size. 

The  biological  differences  on  agar-agar,  blood  serum, 
gelatin,  and  bouillon,  between  the  members  of  the  inter- 
mediate group,  and  between  bacillus  typhosus  and  bacillus 
coli  are  too  insignificant  and  uncertain  to  be  of  any  assist- 
ance in  a  differentiation  between  members  of  the  group. 
In  litmus  milk  certain  well-marked  differences  between 
different  members  of  the  group  are  noticed.  None  of  the 
organisms  of  the  intermediate-  group  produce  coagulation. 
Some  produce  a  slight  initial  acidity,  which  is  later  followed 
by  an  alkaline  reaction.  Still  other  members  of  the  group 
produce  an  acidity  amounting  to  1  per  cent. 

Buxton  states  that  the  intermediates  can  be  distinguished 
from  bacillus  typhosus  by  their  power  of  fermenting  the 
disaccharid  maltose  and  all  the  monosaccharids  with  gas 
formation.  On  the  other  hand  they  can  be  distinguished 
from  bacillus  coli  by  their  inability  to  form  acid  and  gas  in 
lactose  media. 

The  agglutination  reaction  of  members  of  the  intermediate 
group  with  the  serum  of  an  animal  immunized  with  one  of 


540     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

the  organisms  varies  with  the  different  strains.  The  more 
closely  a  member  of  the  group  resembles  culturally  the 
organism  employed  in  immunizing  the  animal  the  more 
readily  is  it  agglutinated.  In  attempts  to  diagnose  para- 
typhoid infection  it  is  well  to  bear  this  fact  in  mind  and  make 
agglutination  tests  upon  different  members  of  the  group 
with  the  blood  of  the  patient. 


CHAPTER  XXV. 


The  Group  of  Bacilli  Found  in  Cases  of  Epidemic,  Endemic,  and  Sporadic 
Dysentery — The  Morphological,  Biological,  and  Pathogenic  Char- 
acters of  the  Several  Members  of  the  Group — The  Differentiation  of 
the  Different  Types  of  Bacilli. 


BACILLUS   DYSENTERIC. 

THE  investigations  of  epidemic  dysentery  by  Shiga, 
Flexner,  Kruse,  Vedder,  Duval,  Basset,  Park,  and  many 
others,  have  demonstrated  that  this  disease  is  caused  by 
an  organism  that  varies  somewhat  in  its  characters  as 
ericountered  in  different  cases.  So  far  at  least  four  types  of 
organisms  have  been  found  that  differ  in  minor  particulars. 
The  type  of  organism  first  encountered  by  Shiga,  in  Japan, 
is  the  one  that  is  probably  very  widely  distributed,  because 
it  has  been  found  in  practically  every  place  where  search 
has  been  made  for  it.  The  type  of  organism  encountered 
by  Flexner  in  the  Philippine  Islands,  and  believed  by  him 
to  differ  from  the  Shiga  type,  has  also  been  found  very 
generally  in  the  United  States,  especially  in  dysentery 
occurring  in  infants.  The  type  of  organism  isolated  by 
Hiss  and  Russell,  and  later  by  Park  and  his  associates,  has 
most  of  the  characteristics  of  the  Flexner  type  of  organism, 
though  the  agglutination  reaction  shows  that  it  is  not 
identical  with  it. 

At  first  certain  investigators  were  inclined  to  regard  the 
Flexner  type  of  organism  as  having  no  causative  relation 
(541) 


542      APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

whatever  to  dysentery,  but  later  detailed  studies  all 
strengthen  the  assumption  that  the  Shiga  type  of  the 
organism  is  not  the  only  one  concerned  in  causing  epidemic 
dysentery.  In  a  number  of  cases  of  dysentery  two,  and  at 
times  three,  types  of  bacillus  dysenterise  have  been  encoun- 
tered. Thus  far  it  has  been  impossible  to  differentiate  clini- 
cally between  the  infections  produced  by  the  one  or  the 
other  type,  both  severe  and  mild  cases  being  caused  by  each. 

The  Shiga  Type  of  Organism. — The  evidence  presented 
by  Shiga,  who  discovered  this  organism  in  1898,  in  Japan, 
and  the  subsequent  observations  of  Flexner  upon  dysentery 
in  the  Philippine  Islands,  leaves  little  room  for  doubt  that, 
in  so  far  as  acute  epidemic  dysentery  is  concerned,  the 
organism  under  consideration  may  reasonably  be  regarded  as 
the  causative  factor.  By  both  Shiga  and  Flexner  the 
organism  was  almost  uniformly  encountered  in  the  intestinal 
contents,  the  intestinal  walls,  and  the  mesenteric  glands 
during  the  acute  stages  of  the  disease.  Later  it  was  fre- 
quently missed,  and  this  became  more  common  as  the 
malady  progressed  to  chronicity  or  recovery. 

It  is  a  bacillus  of  medium  size,  with  rounded  ends.  In 
general  its  morphology  may  properly  be  likened  to  that  of 
either  the  typhoid  or  colon  bacillus. 

It  is  motile  and  does  not  form  spores. 

.  It  can  be  stained  with  any  of  the  ordinary  aniline  dyes. 
It  is  decolorized  by  the  method  of  Gram.  It  may  be  cul- 
tivated on  all  the  ordinary  media.  It  grows  at  room- 
temperature,  but  better  at  the  temperature  of  the  body. 
It  does  not  liquefy  gelatin. 

The  colonies  upon  agar-agar  present  nothing  character- 
istic; those  on  gelatin  are  at  first— i.  e.,  just  after  isolation 
from  the  body — like  those  of  bacillus  typhosus;  later  on, 


BACILLUS  DYSENTERIC  543 

after  the  organism  has  been  kept  under  conditions  of  con- 
tinuous saprophytic  growth,  the  colonies  may  be  thicker, 
denser,  moister,  and  less  translucent,  but  always  suggesting 
the  peculiar,  leaf-like  contour  characteristic  of  the  colonies 
of  the  colon-typhoid  group  under  similar  conditions.  In 
gelatin  stab-cultures  there  is  growth  along  the  track  made 
by  the  needle,  and  little  tendency  to  lateral  development 
over  the  surface. 

On  potato,  its  growth  may  be  so  limited  as  to  be  scarcely 
visible,  or  it  may  appear  as  a  moderately  voluminous  gray- 
ish-brown or  light-brown  layer  along  the  track  made  by 
the  needle,  and  spreading  laterally  beyond  this.  Between 
these  extremes  all  gradations  'may  be  seen  according  to 
the  suitability  of  the  potato  used. 

In  bouillon  it  causes  uniform  clouding  and  a  more  or  less 
dense  sediment.  It  does  not  form  a  pellicle. 

Growth  on  blood-serum  is  not  accompanied  by  liquefac- 
tion (digestion). 

Glycerin-agar-agar  appears  less  suited  to  its  growth  than 
plain  nutrient  agar-agar. 

It  does  not  ferment  either  glucose,  saccharose,  or  lactose, 
with  liberation  of  gas;  although  in  glucose  media  there  is 
a  slight  increase  of  acidity. 

When  grown  in  litmus-milk,  the  latter,  after  twenty-four 
to  seventy-two  hours  at  body-temperature,  becomes  a  pale 
lilac.  Later  on — i.  e.,  after  six  to  eight  days — there  is  a 
development  of  alkali,  and  the  lilac  tint  gives  way  to  a 
deep,  distinct  blue  color.  Coagulation  is  never  observed. 

It  is  either  incapable  of  producing  indol,  or  has  this 
faculty  developed  to  so  limited  a  degree  as  to  make  the 
matter  doubtful. 

When  mixed  with  blood  serum  of  individuals  suffering 


544     APPLICATION  OF  METHODS  OF   BACTERIOLOGY 

from  this  form  of  dysentery  a  positive  agglutination  reaction 
is  often  obtained. 

It  is  pathogenic  by  both  subcutaneous  and  intraperitoneal 
inoculation  for  the  ordinary  laboratory  test-animals— i.  e.t 
mice,  guinea-pigs,  and  rabbits. 

When  injection  is  made  beneath  the  skin,  death  results 
in  from  two  to  four  days,  according  to  the  dose  and  viru- 
lence .of  the  culture  used. 

The  most  striking  lesion  is  that  observed  at  and  about 
the  site  of  inoculation.  This  consists  of  edema,  hemor- 
rhagic  exudation,  and  in  delayed  cases,  more  or  less  of  pus 
formation.  The  subcutaneous  lymph-glands  are  often  en- 
larged and  reddened,  and  a  serous  exudation  is  frequently 
encountered  in  the  great  serous  cavities.  Of  the  animals 
mentioned,  the  rabbit  is  most  apt  to  survive  the  subcu- 
taneous inoculation. 

When  injected  into  the  peritoneal  cavity,  death  takes 
place  in  from  a  few  hours  to  five  or  six  days,  according 
to  dose  and  virulence  of  the  culture  used. 

At  autopsy  the  superficial  lymph-glands  are  enlarged  and 
reddened;  the  peritoneum  contains  more  or  less  of  turbid 
fluid  and  small  masses  of  leukocytes;  the  pleural  and  peri- 
cardial  cavities  may  contain  clear  fluid;  the  spleen  is  swollen; 
the  adrenals  and  kidneys  are  congested;  there  may  be  a 
grayish  exudate  over  the  liver,  spleen,  and  intestines,  the 
bloodvessels  are  injected  ?  the  small  intestine  may  be  filled 
with  semifluid  or  fluid  matter;  there  may  be  ecchymosis 
in  the  intestinal  mucosa,  and  Peyer's  patches  may  be 
enlarged  and  reddened. 

The  distribution  of  the  bacilli  varies :  sometimes  there 
is  a  general  invasion  of  the  body  by  the  bacilli;  at  others 
they  are  only  to  be  found  at  the  local  site  of  inoculation. 


BACILLUS  DYSENTERIC  545 

Sometimes  they  can  be  detected  in  the  intestinal  contents 
after  both  subcutaneous  and  intraperitoneal  inoculation; 
at  other  times  they  cannot. 

If  the  stomach  contents  be  neutralized  and  large  doses  of 
the  bacilli  be  administered  per  os,  death  may  occur.  Under 
these  conditions  the  small  intestine  is  hyperemic  and  con- 
tains blood-stained  mucoid  matter,  from  which  the  bacilli 
may  usually  be  cultivated. 

If  cultures  be  fed  to  cats  after  administration  of  croton 
oil,  a  fatal  diarrhea  may  ensue.  The  mucous  membrane  of 
the  large  intestine  is  injected,  its  surface  covered  with 
mucous,  and  its  contents  mucoid.  From  the  latter  the 
bacilli  may  be  recovered  in  culture. 

A  fatal  diarrhea  may  follow  the  simple  feeding  of  cultures 
to  dogs.  This  occurs  in  somewhat  less  than  six  days.  The 
condition  of  the  contents  and  walls  of  the  large  intestine 
is  essentially  similar  to  that  seen  in  the  cat. 

In  view  of  the  fact  that  marked  evidences  of  intoxica- 
tion may  follow  upon  the  injection  of  suspensions  of  dead 
cultures  of  this  organism  (solid  cultures  killed  by  exposure 
to  60°  C.),  it  is  probable  that  the  pathogenicity  of  this 
organism  is  referable  to  its  endotoxin,  rather  than  to  a 
soluble  intoxicant  secreted  or  manufactured  as  a  by  product 
in  the  course  of  growth. 

The  Hiss-Russell  Type  of  Organism. — In  the  detailed  study 
of  dysentery  and  summer  diarrhea  in  infants,  a  type  of  bacil- 
lus dysenterise  has  been  encountered  which  has  the  property 
of  fermenting  mannite  as  well  as  dextrose.  The  Shiga  type 
ferments  dextrose,  but  none  of  the  other  carbohydrates. 

The  Strong  Type  of  Organism. — This  type  of  organism  has 
many  of  the  characters  of  the  Harris  type,  though  it  ferments 
only  mannito-dextrose,  and  saccharose. 
35 


546      APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

The  Harris  Type  of  Organism. — This  type  of  bacillus 
dysenterise  was  first  encountered  by  Strong  while  working 
in  the  Philippine  Islands.  It  has  since  been  encountered 
quite  frequently  in  the  United  States,  especially  in  the 
summer  diarrheas  in  infants.  This  organism  ferments 
mannite  as  well  as  dextrose,  maltose,  saccharose,  and 
dextrin. 

It  is  only  by  careful  observations  of  the  reactions  with 
the  different  carbohydrates  that  it  is  possible  by  culture 
methods  to  differentiate  between  these  different  strains 
of  bacillus  dysenterise,  as  has  been  shown  by  Hiss1  and  by 
others. 

The  Agglutinability  of  Bacillus  Dysenterise. — The  influence 
of  agglutinins  in  dysentery  immune  serum  has  also  served 
to  differentiate  between  different  types  of  baccillus  dysen- 
terise. Normal  serums,  especially  those  of  bovines  and 
of  goats,  also  yield  very  instructive  results.  Variations 
in  the  agglutinability  of  the  several  types  of  bacillus 
dysenterise,  especially  in  normal  serums,  were  first  pointed 
out  by  Bergey,2  and  have  since  been  noticed  by  other 
investigators  (see  especially  Park  and  Hiss,  loc.  cit.). 

The  different  types  of  bacillus  dysenterise  can  be  distin- 
guished easily  by  their  relative  agglutinability,  but  in  order 
to  do  so  animals  must  be  rendered  immune  from  each 
variety  and  the  serum  of  such  animals  employed  as  specific 
reagents.  When  this  is  done  it  will  be  found  that  the  serum 
of  an  animal  immunized  with  the  Shiga  type  of  organism 
will  agglutinate  that  type  of  organism  in  high  dilutions, 
say  1  :  5000,  while  the  Harris  type  of  organism  will  only  be 
agglutinated  in  dilutions  of  1  :  200,  and  the  Hiss-Russell 

1  Journal  of  Medical  Research,  December,  1904,  viii. 

2  Ibid.,  1903,  v,  21. 


BACILLUS  DYSENTERIC  547 

type  of  organism  in  dilutions  of  1  : 50.  On  the  other  hand, 
the  serum  of  an  animal  immunized  with  the  Flexner  type 
of  organism  will  agglutinate  that  type  of  organism  in  high 
dilutions,  say  1  :  10,000,  while  the  other  two  types  of  the 
organism  will  be  agglutinated  only  in  dilutions  of  1  : 100. 
The  serum  of  an  animal  immunized  with  the  Hiss-Russell 
type  of  organism  will  agglutinate  that  type  of  organism 
in  dilutions,  say  of  1  : 1000,  while  the  Harris  type  is  agglu- 
tinated only  in  dilutions  of  1  : 100,  and  the  Shiga  type  in 
dilutions  of  1  :  20. 

Protective  Inoculation. — By  the  repeated  inoculation  of 
animals  with  cultures  of  this  organism,  killed  either  by  heat 
or  by  chemicals,  it  has  been  found  possible  to  protect  them 
against  otherwise  fatal  doses  of  the  living  virulent  organism. 
When  treated  in  this  way,  the  goat  supplies  a  serum  that 
exhibits  not  only  an  agglutinating  power  over  the  living 
bacilli,  but  possesses  both  protective  and  curative  properties 
when  injected  into  other  susceptible  animals. 

During  1898-1899  Shiga1  employed  a  protective  serum, 
made  after  the  foregoing  principles,  in  the  treatment  of 
dysentery  in  human  beings.  During  the  period  mentioned 
he  treated  266  cases,  and  had  a  death-rate  of  9.6  per  cent.; 
while  for  1736  cases  occurring  at  the  same  time  and  in  the 
same  locality,  but  not  so  treated,  there  was  a  death-rate 
of  34.7  per  cent.2 

Holt3  summarizes  the  results  obtained  in  the  treatment 

1  See  The  Epidemic  Dysentery  of  the  Past  Twenty  Years  in  Japan,  by 
Stuart   Eldridge,    M.D.,    U.    S.    Marine-Hospital    Service,    Public   Health 
Reports,  1900,  xv,  No.  1,  1-11. 

2  The  foregoing  sketch  is  compiled  from: 

Shiga,  Ueber  den  Dysenteric-bacillus  (Bacillus  dysenteriae) ,  Centralblatt 
fur  Bakteriologie  und  Parasitenkunde,  1898,  Abt.  i,  Bd.  xxiv,  Nos.  22,  23,  24. 

Flexner,  On  the  Etiology  of  Tropical  Dysentery,  Philadelphia  Medical 
Journal,  September  1,  1900. 

3  Studies  from  the  Rockefeller  Institute  for  Medical  Research,  1904,  vol.  ii. 


548     APPLICATION  OF  METHODS   OF   BACTERIOLOGY 

of  87  cases  with  dysentery  immune  serum.  Decided  im- 
provement was  noted  in  only  12  of  the  patients.  These 
were  principally  hospital  cases,  and  hence  rather  grave 
forms  of  the  disease.  Another  factor  which  probably 
operated  against  the  favorable  influence  of  the  serum  is  the 
fact  that  the  serum  treatment  was  generally  preceded  by 
a  careful  bacteriological  'analysis  of  the  stools  in  order  to 
establish  a  positive  diagnosis,  requiring  two  or  three  days 
so  that  the  serum  treatment  was  instituted  late  in  the  course 
of  the  disease. 

Holt  points  out  that  the  conditions  necessary  to  obtain 
success  in  the  serum  treatment  of  cases  of  dysentery  are: 
First,  the  early  use  of  the  serum,  before  serious  lesions  have 
developed  or  before  the  patient's  general  condition  has  been 
too  profoundly  impaired;  second,  the  serum  must  be 
administered  in  repeated  doses,  one  or  two  doses  a  day,  and 
continued  for  several  days  in  severe  cases. 


CHAPTER  XXVI. 

The  Spirillum  (Comma  Bacillus)  of  Asiatic  Cholera — Its  Morphologica 
and  Cultural  Peculiarities — Pathogenic  Properties — The  Bacterio- 
logical Diagnosis  of  Asiatic  Cholera — Microspira  Metchnikovi — Micro- 
spira  ("Vibrio")  Schuylkilliensis — Its  Morphological,  Cultural,  and 
Pathogenic  Characters. 


THE  CHOLERA  GROUP  OF  ORGANISMS. 

AT  the  conference  held  in  Berlin  in  1884  for  the  purpose 
of  discussing  Asiatic  cholera  from  the  sanitary  aspect,  it 
was  announced  by  Koch1  that  he  had  discovered  in  the 
intestinal  evacuations  of  individuals  suffering  from  Asiatic 
cholera  a  microorganism  that  he  believed  to  be  the  cause 
of  the  malady.  The  importance  of  this  statement  naturally 
attracted  widespread  attention  to  the  subject,  and  as  one 
of  the  consequences  there  existed,  for  a  short  time  following, 
some  skepticism  as  to  the  accuracy  of  Koch's  claim.  These 
doubts  arose  as  a  result  of  a  series  of  contributions  from 
other  observers,  who  endeavored  to  prove  that  the  organism 
found  by  Koch  in  cholera  evacuations  was  common  to  other 
localities,  and  was  not  a  specific  accompaniment  of  this 
disease.  It  was  not  very  long,  however,  before  it  was 
evident  that  these  objections  were  based  upon  untrust- 
worthy observations,  and  that  by  reliable  methods  of 
investigation  the  organism  to  which  he  had  called  attention 
could  be  easily  differentiated  from  each  of  those  with  which 
it  was  claimed  to  be  identical. 

1  Verhandlungen  der  Conferenz  zur  Erorterung  der  Cholerafrage,  1884, 
Berlin. 

(549) 


550     APPLICATION  OF  METHODS  OF   BACTERIOLOGY 

This  organism,  commonly  known  both  as  the  spirillum 
of  Asiatic  cholera,  and,  because  of  its  morphology,  as  Koch's 
"comma  bacillus/'  is  identified  by  the  following  peculiarities: 

MICROSPIRA  COMMA  (KOCH),  SCHROTER,  1886. 

SYNONYMS.  Comma-bacillus,  Koch,  1884;  Spirillum  cholerae  Asiatica, 
Fliigge,  1886. 

Morphology. — It  is  a  slightly  curved  rod,  ranging  from 
about  0.8  to  2fj.  in  length  and  from  0.3  to  0.4^  in  thickness 
— that  is  to  say,  it  is  usually  from  about  one-half  to  two- 
thirds  the  length  of  the  tubercle  bacillus,  but  is  thicker  and 
plumper.  Its  curve  is  frequently  not  more  marked  than 
that  of  a  comma,  and,  indeed,  it  is  often  almost  straight; 
at  times,  though,  the  curve  is  much  more  pronounced,  and 
may  even  describe  a  semicircle.  Occasionally  the  curve 
may  be  double,  one  comma  joining  another,  with  their 
convexities  pointing  in  opposite  directions,  so  that  a  figure 
similar  to  the  letter  S  is  produced.  In  cultures  long  spiral 
or  undulating  threads  may  often  be  seen.  From  these 
appearances  this  organism  cannot  be  considered  as  a  bacillus, 
but  rather  as  an  intermediate  type  between  the  bacilli  and 
the  spirilla.  Koch  thinks  it  not  improbable  that  the  short 
comma  forms  represent  segments  of  a  true  spirillum,  the 
normal  form  of  the  organism.  (Fig.  86.) 

It  does  not  form  spores,  and  we  have  no  reliable  evidence 
that  it  possesses  the  property  of  entering,  at  any  time,  a 
stage  in  which  its  powers  of  resistance  to  detrimental  agencies 
are  increased. 

It  is  a  flagellated  organism,  but  has  only  a  single  flagellum 
attached  to  one  of  its  ends. 


MICROSPIRA  COMMA  551 

It  is  actively  motile,  especially  in  the  comma  stage 
though  the  long  spiral  forms  also  possess  this  property. 

Grouping. — As  found  in  the  slimy  flakes  in  the  intes- 
tinal discharges  from  cholera  patients,  Koch  likens  its  mode 

FIG.  88 


\- 


Microspira  comma.    Impression  cover-slip  from  a  colony  thirty-four 
hours  old. 


of  grouping  to  that  seen  in  a  school  of  small  fish  when 
swimming  up  stream — i.  e.,  they  all  point  in  nearly  the 

FIG.  89 

/;/  . 

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(>(>  '<••£. 

€    -    ^  '<   vv 
Involution-forms  of  microspira  comma,  as  seen  in  old  cultures. 

same  direction,  and  lie  in  irregularly  parallel,  linear  groups 
that  are  formed  by  one  comma  being  behind  the  other  with- 
out being  attached  to  it. 
On  cover-slip  preparations  made  from  cultures  in  the 


552      APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

ordinary  way  there  is  nothing  characteristic  about  the 
grouping;  but  in  impression  cover-slips  made  from  young 
cultures  the  short  commas  will  nearly  always  be  seen  in 
small  groups  of  three  or  four,  lying  together  in  such  a  way 
as  to  have  their  long  axes  nearly  parallel  to  one  another. 
(See  Fig.  88.) 

In  old  cultures  in  which  development  has  ceased  it  under- 
goes degenerative  changes,  and  the  characteristic  comma 
and  spiral  shapes  may  entirely  disappear,  their  place  being 
taken  by  irregular  involution-forms  that  present  every 
variety  of  outline.  (See  Fig.  89.)  In  this  stage  they  take 
on  the  stain  very  feebly,  and  often  not  at  all. 

Cultural  Peculiarities. — On  plates  of  nutrient  gelatin  that 
have  been  prepared  from  a  pure  culture  of  this  organism 
and  kept  at  a  temperature  of  from  20°  to  22°  C.,  develop- 
ment can  often  be  observed  after  as  short  a  period  as  twelve 
hours,  but  frequently  not  before  sixteen  to  eighteen  hours. 
This  is  especially  true  of  the  first  or  "original"  plate,  con- 
taining the  largest  number  of  colonies.  At  this  time  the 
plate  will  present  to  the  naked  eye  an  appearance  that  has 
been  likened  to  a  ground-glass  surface,  or  to  a  surface  that 
has  been  stippled  with  a  finely  pointed  needle,  or  one  upon 
which  very  fine  dust  has  been  sprinkled.  This  appearance 
is  due  to  the  presence  of  minute  colonies  closely  packed 
together  upon  the  surface  of  the  gelatin.  In  the  depth 
of  the  gelatin  can  also  be  seen  closely  packed,  small  points, 
likewise  representing  growing  colonies.  As  growth  progresses 
liquefaction  occurs  around  the  superficial  colonies,  and  in 
consequence  this  plate  is  usually  entirely  liquid  after  from 
twenty-four  to  thirty  hours;  the  developmental  phases 
through  which  the  colonies  pass  cannot,  therefore,  be  studied 
upon  it. 


MICROSPIRA  COMMA  553 

On  plates  2  and  3,  where  the  colonies  are  more  widely 
separated,  they  can  be  seen  after  twenty-four  to  thirty 
hours  as  small,  round  or  oval,  white  or  cream-white  points, 
and  when  located  superficially  a  narrow  transparent  zone 
of  liquefaction  can  be  detected  around  them.  As  growth 
continues  this  liquefaction  extends  downward  rather  than 
laterally,  and  the  colony  ultimately  assumes  the  appearance 
of  a  dense,  white  mass  lying  at  the  bottom  of  a  sharply-cut 

FIG.  90 


Development  phases  of  colonies  of  microspira  comma  at  20°  to  22°  C.  on 
gelatin.  X  about  75  diameters,  a,  after  sixteen  to  eighteen  hours;  6,  after 
twenty-four  to  twenty-six  hours;  c,  after  thirty-eight  to  forty  hours;  d,  after 
forty-eight  to  fifty  hours;  e,  after  sixty-four  to  seventy  hours. 


pit  or  funnel  containing  transparent  fluid.  This  liquefaction 
is  never  very  widespread  nor  rapid,  and  rarely  extends 
more  than  one  millimeter  beyond  the  colony  proper.  On 
plates  containing  few  colonies  there  is  little  or  no  tendency 
for  them  to  become  confluent,  and  they  rarely  exceed  2 
to  3  mm.  in  diameter. 

When  examined  under  a  low  magnifying  lens  the  very 
young  colonies  (sixteen  to  eighteen  hours  old)  appear  as 
pale,  translucent,  granular  globules  of  a  very  delicate 


554     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

greenish  or  yellowish-green  color,  sharply  outlined,  and 
not  perfectly  round.  (See  a,  Fig.  90.)  As  growth  progresses 
this  homogeneous  granular  appearance  is  replaced  by  an 
irregular  lobulation,  and  ultimately  the  sharply-cut  margin 
of  the  colony  becomes  dentated  or  scalloped.  (See  b  and  c, 
Fig.  90.)  After  forty-eight  hours  (and  frequently  sooner) 
liquefaction  of  the  gelatin  has  taken  place  to  such  an  extent 
that  the  appearance  of  the  colony  is  entirely  altered.  Under 
a  magnifying  glass  the  colony  proper  is  now  seen  to  be 
ragged  about  its  edges,  while  here  and  there  shreds  of  the 
colony  can.be  detected  scattered  through  the  liquid  into 
which  it  is  sinking.  These  shreds  evidently  represent 
portions  of  the  colony  that  became  detached  from  its  margin 
as  it  gradually  sank  into  the  liquefied  area. 

At  d,  in  Fig.  90,  is  seen  a  representation  of  the  several 
appearances  afforded  by  the  colonies  at  this  stage.  At  the 
end  of  the  second,  or  during  the  early  part  of  the  third  day, 
the  sinking  of  the  colonies  into  the  liquefied  pits  resulting 
from  their  growth  is  about  complete,  and  under  a  low-power 
lens  they  now  appear  as  dense,  granular  masses,  surrounded 
by  an  area  of  liquefaction  through  which  can  be  seen 
granular  prolongations  of  the  colony,  usually  extending 
irregularly  between  the  periphery  and  the  central  mass. 
(See  e,  Fig.  90.)  If  the  periphery  be  examined,  it  will  be  seen 
to  be  fringed  with  delicate,  cilia-like  lines  that  radiate  from 
it  in  much  the  same  way  that  cilia  radiate  from  the  ends 
of  the  columnar  epithelial  cells  lining  the  air-passages. 

These  are  the  more  marked  phases  through  which  the 
colonies  of  this  organism  pass  in  their  development  on 
gelatin  plates.  In  some  cultures  the  various  phases  here 
given  pass  in  succession  more  quickly,  while  in  cultures 
from  other  sources  they  may  be  somewhat  retarded. 


MICROSPIRA   COMMA 


555 


On  plates  of  nutrient  agar-agar  the  appearance  of  the 
colonies  is  not  characteristic.  They  appear  as  round  or 
oval  patches  of  growth  that  are  moist  and  moderately 
transparent.  The  colonies  on  this  medium  at  37°  C.  natu- 
rally grow  to  a  larger  size  than  do  those  upon  gelatin  at 
22°  C.  tl^ 

FIG.  91  j 


a 


abed 
Stab-culture  of  microspira  comma  in  gelatin,  at  18°  to  20°  C.     a,  after 
twenty-four  hours;  b,  after  forty-eight  hours;  c,  after  seventy- two  hours; 
d,  after  ninety-six  hours. 

In  stab-cultures  in  gelatin  there  appears  at  the  top  of 
of  the  needle-track  after  thirty-six  to  forty-eight  hours 
at  22°  C.  a  small,  funnel-shaped  depression.  As  the  growth 
progresses  liquefaction  occurs  about  this  point.  In  the 
center  of  the  depression  can  be  distinguished  a  small,  dense, 


556      APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

whitish  clump,  the  colony  itself.  As  growth  continues  the 
.  depression  increases  in  extent  and  ultimately  assumes  an 
appearance  that  consists  in  the  apparent  sinking  of  the 
liquefied  portion  in  such  a  way  as  to  leave  a  perceptible 
air-space  between  the  top  of  the  liquid  and  the  surface  of 
the  solid  gelatin.  The  growth  now  appears  to  be  capped  by 
a  small  air-bubble.  The  impression  given  by  it  at  this 
stage  is  not  only  that  there  has  been  a  liquefaction,  but 
also  a  coincident  evaporation  of  the  fluid  from  the  liquefied 
area  and  a  constriction  of  the  superficial  opening  of  the 
funnel.  (See  a,  b,  c,  and  d,  Fig.  91.)  Liquefaction  is  not 
especially  active  along  the  deeper  portions  of  the  track 
made  by  the  needle,  though  in  stab-cultures  in  gelatin  the 
liquefaction  is  much  more  extensive  than  that  usually  seen 
around  colonies  on  plates.  It  spreads  laterally  at  the  upper 
portion,  and  after  about  a  week  a  large  part  of  the  gelatin 
in  the  tube  may  have  become  fluid,  and  the  growth  will 
have  lost  its  characteristic  appearance. 

Stab-  and  smear-cultures  on  agar-agar  present  nothing 
characteristic. 

Its  growth  in  bouillon  is  luxuriant,  causing  a  diffuse 
clouding  and  the  ultimate  production  of  a  delicate  film 
upon  the  surface. 

In  sterilized  milk  of  a  neutral  or  amphoteric  reaction  at 
a  temperature  of  36°  to  38°  C.  it  develops  actively,  and 
gradually  produces  an  acid  reaction,  with  coagulation  of 
the  casein.  It  retains  its  vitality  under  these  conditions 
for  about  three  weeks  or  more.  The  blue  color  of  milk  to 
which  neutral  litmus  tincture  has  been  added  is  changed  to 
pink  after  thirty-six  or  forty-eight  hours  at  body-temperature. 

Its  growth  in  peptone  solution,  either  that  of  Dunham 
(see  Special  Media)  or  the  one  preferred  by  Koch,  viz., 


MICROSPIRA   COMMA  557 

2  parts  of  Witte's  peptone,  1  part  of  sodium  chloride,  and 
100  parts  of  distilled  water,  is  accompanied  by  the  produc- 
tion of  both  indol  and  nitrites,  so  that  after  eight  to  twelve 
hours  in  the  incubator  at  37°  C.  the  rose  color  characteristic 
of  indol  appears  upon  the  addition  of  sulphuric  acid  alone. 
(See  Indol  Reaction.) 

(What  does  the  presence  of  nitrites  in  these  cultures 
signify?) 

In  peptone  solution  to  which  rosolic  acid  has  been  added 
the  red  color  is  very  much  intensified  after  four  or  five  days 
at  37°  C. 

Its  growth  on  potato  of  slightly  acid  reaction  is  seen  after 
three  or  four  days  at  37°  C.  as  a  dull,  whitish,  non-glistening 
patch  at  and  about  the  site  of  inoculation.  It  is  not  elevated 
above  the  surface  of  the  potato,  and  can  only  be  distinctly 
seen  when  held  to  the  light  in  a  particular  position.  Growth 
on  acid  potato  occurs,  however,  only  at  or  near  the  body- 
temperature,  owing  probably  to  the  acid  reaction,  which 
is  sufficient  to  prevent  development  at  a  lower  temperature, 
but  does  not  have  this  effect  when  the  temperature  is  more 
favorable.  On  solidified  blood-serum  growth  is  usually 
said  to  be  accompanied  by  slow  liquefaction.  I  have  not 
succeeded  in  obtaining  this  result  on  Loffler's  serum,  nor 
have  I  detected  anything  characteristic  about  its  growth  on 
this  medium. 

The  temperature  most  favorable  for  its  growth  is  between 
35°  and  38°  C.  It  grows,  but  more  slowly,  at  17°  C.  Below 
16°  C.  no  growth  is  visible. 

It  is  not  destroyed  by  freezing.  When  exposed  to  65°  C. 
its  vitality  is  destroyed  in  five  minutes. 

It  is  strictly  aerobic,  its  development  ceasing  if  the  supply 
of  oxygen  be  cut  off. 


558     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

It  does  not  grow  in  an  atmosphere  of  carbonic  acid,  but 
is  not  killed  by  a  temporary  exposure  to  this  gas.  It  does 
not  grow  in  acid  media,  but  flourishes  best  in  media  of 
neutral  or  slightly  alkaline  reaction.  It  is  so  sensitive  to 
the  action  of  acids  that  at  22°  C.  its  development  is  arrested 
when  an  acid  reaction  equivalent  to  0.066  to  0.08  per  cent, 
of  hydrochloric  or  nitric  acid  is  present.  (Kitasato.) 

Under  artificial  cultivation  the  maximum  development 
of  this  organism  is  reached  in  a  comparatively  short  time; 
after  this  it  remains  quiescent  for  a  period,  and  finally 
degeneration  or  involution  begins.  When  in  this  state 
they  take  up  coloring  reagents  very  faintly  or  not  at  all, 
and  may  lose  entirely  their  characteristic  shape.  (See  Fig. 
93.) 

When  present  with  other  bacteria,  under  conditions 
favorable  to  growth,  the  comma  bacillus  at  first  grows 
much  more  rapidly  than  do  the  others;  in  twenty-four 
hours  it  will  often  so  outnumber  the  other  organisms  present 
that  microscopic  examination  might  lead  one  to  regard  the 
material  under  consideration  as  a  pure  culture  of  this 
organism.  Its  conspicuous  development  under  these  cir- 
cumstances does  not,  however,  last  longer  than  two  or 
three  days;  degeneration  and  death  begin,  and  the  other 
organisms  gain  the  ascendancy.  This  fact  has  been  taken 
advantage  of  in  the  bacteriological  diagnosis  of  cholera. 

In  connection  with  his  experiments  upon  the  poison 
produced  by  the  cholera  organism  Pfeiffer1  states  that  in 
very  young  cultures,  grown  under  access  to  oxygen,  there 
is  present  a  body  that  possesses  intensely  toxic  properties. 
This  primary  cholera-poison  stands  in  very  close  relation 
to  the  material  composing  the  bodies  of  the  bacteria  them- 

1  Zeitschrift  fur  Hygiene  und  Infektionskrankheiten,  Bd.  xi,  S.  393. 


MICROSPIRA  COMMA  559 

selves,  and  is  probably  an  integral  constituent  of  them,  for 
the  vitality  of  the  cholera  spirilla  can  be  destroyed  by  means 
of  chloroform  or  thymol,  or  by  drying,  without  apparently 
any  alteration  of  this  poisonous  body.  Absolute  alcohol, 
concentrated  solutions  of  neutral  salts,  and  a  temperature 
of  100°  C.,  decompose  this  substance,  leaving  intact  second- 
ary poisons  which  possess  a  similar  physiological  activity, 
but  only  when  given  in  from  ten  to  twenty  times  the  dose 
necessary  to  produce  the  same  effects  with  the  primary 
poison. 

Experiments  upon  Animals. — As  a  result  of  experiments 
for  the  purpose  of  determining  if  the  disease  can  be  pro- 
duced in  any  of  the  lower  animals  it  has  been  found  that 
white  mice,  monkeys,  cats,  dogs,  poultry,  and  many  other 
animals  are  not  susceptible  to  infection  by  the  methods 
usually  employed  in  inoculation  experiments.  When  animals 
are  fed  on  pure  cultures  of  the  comma  bacillus  no  effect 
is  produced,  and  the  organisms  cannot  be  obtained  from 
the  stomach  or  intestines.  They  are  destroyed  in  the 
stomach,  and  do  not  reach  the  intestines;  they  are  not 
demonstrable  in  the  feces  of  these  animals.  Intra vascular 
injections  of  a  pure  culture  into  rabbits  are  followed  by  an 
illness,  from  which  the  animals  usually  recover  in  from  two 
to  three  days;  intraperitoneal  injections  into  white  mice 
are,  as  a  rule,  followed  by  death  in  from  twenty-four  to 
forty-eight  hours,  the  conditions  in  both  instances  most 
probably  resulting  from  the  toxic  activities  of  the  specific 
poisons  contained  in  the  cultures  used. 

None  of  the  lower  animals  suffer  spontaneously  from 
Asiatic  cholera. 

The  failure  to  induce  cholera  in  animals  by  feeding  or 
by  injection  of  cultures  into  the  stomach,  was  shown  by 


560     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

Nicati  and  Rietsch1  to  be  due  to  the  destructive  action  of 
the  acid  gastric  juice  on  the  organisms.  They  showed  that 
if  cultures  of  this  organism  were  introduced  into  the  aliment- 
ary tract  of  certain  animals  in  such  a  manner  that  they 
would  not  be  subjected  to  the  influence  of  the  gastric  juice, 
a  pathological  condition  closely  simulating  cholera  as  it 
occurs  in  man  could  be  produced.  For  this  purpose  the 
common  bile-duct  was  ligated,  after  which  the  cultures 
were  injected  directly  into  the  duodenum.  Such  inter- 
ference with  the  flow  of  bile  lessens  intestinal  peristalsis, 
and  thus  permits  development  of  the  organisms  at  the  point 
at  which  they  are  deposited — that  is,  the  portion  of  the 
intestine  having  an  alkaline  reaction  and  beyond  the  influence 
of  the  acid  stomach-juice. 

By  this  method  Nicati  and  Rietsch,  Van  Ermengem,2 
Koch,3  and  others  were  enabled  to  produce  in  the  animals 
upon  which  they  operated  a  condition  that  was,  if  not 
identical,  at  all  events  very  similar  pathologically  to  that 
seen  in  the  intestines  of  subjects  dead  of  the  disease. 

At  a  subsequent  conference  held  in  Berlin  in  1885  Koch4 
described  the  following  method,  by  means  of  which  he  had 
been  able  to  obtain  a  much  greater  degree  of  constancy 
in  his  efforts  to  produce  cholera  in  lower  animals:  bearing 
in  mind  the  point  made  by  Nicati  and  Rietsch  as  to  the 
effect  produced  by  the  acid  reaction  of  the  gastric  juice, 
this  reaction  was  first  to  be  neutralized  by  injecting  through 
a  soft  catheter  passed  down  the  esophagus  into  the  stomach 
5  c.c.  of  a  5  per  cent,  solution  of  sodium  carbonate.  Ten 

1  Archiv  de  Phys.  norm,  et  path.,  1885,  t.  vi.  3e  ser.     Comptes  rendus, 
xcix,  p.  928;  Revue  de  Hygiene,  1885;  Revue  de  Medecine,  1885,  v. 

2  Recherches    sur    le    Microbe    du    Cholera    Asiatique,  Paris-Bruxelles, 
1885;  Bull,  de  1'Acad.  toy.  de  Med.  de  Belgique,  xviii,  3e  ser. 

3  Loc.  cit.  4  Loc.  cit. 


MICROSPIRA  COMMA  561 

or  fifteen  minutes  later  this  was  to  be  followed  by  the  injec- 
tion into  the  stomach  (also  through  a  soft  catheter)  of 
10  c.c.  of  a  bouillon  culture  of  microspira  comma.  For  the 
purpose  of  arresting  peristalsis  and  permitting  the  bacteria 
to  remain  in  the  stomach  and  upper  part  of  the  duodenum 
for  as  long  a  time  as  possible,  the  animal  was  to  receive, 
immediately  following  the  injection  of  the  culture,  an 
intraperitoneal  injection,  by  means  of  a  hypodermic  syringe, 
of  1  c.c.  of  tincture  of  opium  for  each  200  grams  of  its 
body-weight.  Shortly  after  this  last  injection  deep  narcosis 
sets  in  and  lasts  from  a  half  to  one  hour,  after  which  the 
animal  is  as  lively  as  ever.  Of  35  guinea-pigs  inoculated 
in  this  way  by  Koch,  30  died  of  an  affection  that  was, 
in  general,  very  similar  to  Asiatic  cholera  as  seen  in 
man. 

The  condition  of  those  animals  before  death  is  described 
as  follows:  twenty-four  hours  after  the  operation  the  animal 
appears  unwell;  there  is  loss  of  appetite,  and  the  animal 
remains  quiet  in  its  cage.  On  the  following  day  a  paralytic 
condition  of  the  hind  extremities  appears,  which,  as  the 
day  wears  on,  becomes  more  pronounced;  the  animal  lies 
quite  flat  upon  its  abdomen  or  on  its  side,  with  legs  extended; 
respiration  is  weak  and  prolonged,  and  the  pulsations  of 
the  heart  are  hardly  perceptible;  the  head  and  extremities 
are  cold,  and  the  body-temperature  is  frequently  subnormal. 
The  animal  usually  dies  after  remaining  in  this  condition 
for  a  few  hours. 

At  autopsy  the  small  intestine  is  found  deeply  injected 
and  filled  with  flocculent,  colorless  fluid.  The  stomach  and 
intestines  do  not  contain  solid  masses,  but  fluid;  when 
diarrhea  does  not  occur,  firm  scybala  may  be  detected  in 
the  rectum.  Both  by  microscopic  examination  and  by  cul- 
36 


562     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

ture  methods  the  organisms  are  found  present  in  the  small 
intestine  in  practically  pure  culture. 

Later  Pfeiffer1  determined  that  essentially  similar  con- 
stitutional effects  may  be  produced  in  guinea-pigs  by  the 
intraperitoneal  injection  of  relatively  large  numbers  of  this 
organism.  His  plan  is  to  scrape  from  the  surface  of  a 
fresh  culture  on  agar-agar  as  much  of  the  growth  as  can 
be  held  upon  a  medium  size  wire  loop.  This  is  then  finely 
divided  in  1  c.c.  of  bouillon,  and  by  means  of  a  hypodermic 
syringe  is  injected  directly  into  the  peritoneal  cavity. 
When  virulent  cultures  have  been  used  this  operation  is 
quickly  followed  by  a  fall  in  the  temperature  of  the  animal 
that  is  gradual  and  continuous  until  death  ensues,  which 
usually  occurs  in  from  eighteen  to  twenty-four  hours  after 
the  operation,  though  exceptionally  the  animal  recovers, 
even  after  having  exhibited  marked  symptoms  of  profound 
toxemia. 

Continuing  his  studies  upon  this  disease,  Pfeiffer2  demon- 
strated that  it  is  possible  to  render  an  animal  immune 
from  the  poisonous  properties  of  this  organism  by  repeated 
injections  of  non-fatal  doses  of  dead  cultures  (cultures  that 
have  been  killed  by  the  vapor  of  chloroform  or  by  heat). 
He  also  demonstrated  that  animals  so  immunized  possess 
a  specific  germicidal  action  toward  microspira  comma — i.  e., 
if  into  the  peritoneal  cavity  of  an  animal  immunized  from 
Asiatic  cholera  living  organisms  be  introduced  they  will  all 
be  destroyed  (disintegrated)  within  a  relatively  short  time. 
Furthermore,  if  the  serum  of  an  animal  immunized  from 
cholera  be  injected  into  the  peritoneal  cavity  of  another 
animal  of  the  same  species,  but  not  so  protected,  and  imme- 

1  Zeitschrift  fur  Hygiene  und  Infektionskrankheiten,  Bd.  xi  and  xiv. 

2  Ibid.,  1894,  Bd.  xvii,  S.  355;   1894,  Bd.  xviii,  S.  1;   1895,  Bd.  xx,  S.  197. 


MICROSPIRA   COMMA  563 

diately  afterward  living  cholera  spirilla  be  introduced,  a 
similar  disintegration  and  destruction  of  the  bacteria  will 
also  result.  He  shows  that  a  more  or  less  definite  relation 
exists  between  the  amount  of  serum  and  the  number  of 
organisms  introduced.  Such  a  destruction  *of  microspira 
comma  by  the  serum  of  an  immunized  animal  does  not 
occur  outside  the  animal  body — that  is,  it  cannot  be  demon- 
strated in  a  test-tube,  unless,  as  Bordet  demonstrated,  it  be 
perfectly  fresh  from  the  animal  body,  or,  as  Metchnikoff 
showed,  there  be  added  to  it  a  small  quantity  of  fresh 
serum  from  a  normal  guinea-pig.  The  specificity  of  this 
reaction  is  suggested  by  Pfeiffer  as  a  means  of  differentiating 
the  cholera  spirillum  from  other  suspicious  species,  for  no 
such  bacteriolytic  action  is  observed  if  other  bacteria  be 
introduced  into  the  peritoneal  cavity  of  animals  immunized 
from  Asiatic  cholera. 

Pfeiffer  further  demonstrated  that  the  serum  of  animals 
artificially  immunized  from  Asiatic  cholera  has  an  agglu- 
tinating effect  upon  fluid  cultures  of  microspira  comma 
similar  to  that  seen  when  typhoid  bacilli  are  mixed  with 
serum  from  typhoid  cases,  or  from  animals  artificially 
immunized  from  typhoid  infection  or  intoxication.  (See 
Agglutinin.) 

General  Considerations. — In  all  cases  of  Asiatic  cholera, 
and  only  in  this  disease,  the  organism  just  described  can  be 
detected  in  the  intestinal  evacuations.  The  more  acute  the 
case  and  the  more  promptly  the  examination  is  made  after 
the  evacuations  have  passed  from  the  patient,  the  less 
difficulty  will  be  experienced  in  detecting  the  organism. 

In  some  cases  the  organism  can  be  detected  in  the  vomited 
matters,  though  by  no  means  so  constantly  as  in  the  intes- 
tinal contents. 


564      APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

As  a  rule,  bacteriological  examination  fails  to  reveal  the 
presence  of  the  organisms  in  the  blood  and  internal  organs 
in  this  disease,  though  exceptions  have  been  noted. 

Microspira  comma  is  a  facultative  saprophyte;  that  is 
to  say,  it  apparently  finds  in  certain  parts  of  the  world, 
particularly  in  those  countries  in  which  Asiatic  cholera  is 
endemic,  conditions  that  are  not  entirely  unfavorable  to 
its  development  outside  of  the  body.  This  was  found  to 
be  the  case  not  only  by  Koch,  who  detected  the  presence 
of  the  organism  in  water-tanks  in  India,  but  by  many 
other  observers  who  have  succeeded  in  demonstrating  its 
growth  under  conditions  not  embraced  in  the  ordinary 
methods  employed  for  the  cultivation  of  bacteria.1 

The  results  of  experiments  having  for  their  object  the 
determination  of  the  length  of  time  during  which  the 
organism  may  retain  its  vitality  in  water  are  conspicuous 
for  their  irregularity. 

Koch  states  that  in  ordinary  spring-water  or  well-water 
the  organisms  retained  their  vitality  for  thirty  days,  whereas 
in  the  sewage  of  Berlin  they  died  after  six  or  seven  days; 
but  if  this  latter  were  mixed  with  fecal  matters,  the  organ- 
isms retained  their  vitality  for  but  twenty-seven  hours; 
and  in  the  undiluted  contents  of  cesspools  it  was  impossible 
to  demonstrate  them  after  twelve  hours.  In  the  experi- 
ments of  Nicati  and  Rietsch  they  retained  their  vitality 
in  Marseilles  sewage  for  thirty-eight  days;  in  sea-water, 
sixty-four  days;  in  harbor-water,  eighty-one  days;  and  in 
bilge-water,  thirty-two  days. 

In  one  test  with  the  water-supply  of  Berlin  the  organism 

1  Obviously  all  pathogenic  bacteria  that  have  been  isolated  under  artificial 
methods  of  cultivation  are  facultative  saprophytes.  Were  they  obligate 
parasites,  their  cultivation  upon  dead  materials  would  be  impossible. 


MICROSPIRA  COMMA  565 

retained  its  vitality  for  267  days,  and  in  another  for  382  days, 
notwithstanding  the  fact  that  many  other  organisms  were 
present  at  the  same  time.  There  is  no  ready  explanation 
for  these  variations,  for  they  depend  apparently  upon  a 
number  of  factors  which  may  act  singly  or  together.  For 
example,  in  general  it  may  be  said  that  the  higher  the  tem- 
perature of  the  water  in  which  these  organisms  are  present, 
up  to  20°  C.,  the  longer  do  they  retain  their  vitality;  the 
purer  the  water — that  is,  the  poorer  in  organic  matters — the 
more  quickly  do  the  organisms  die,  whereas  the  richer  it 
is  in  organic  matter  the  longer  do  they  retain  their  vitality. 

The  effect  of  light  upon  growing  bacteria  must  not  be 
lost  sight  of,  for  it  has  been  shown  that  a  surprisingly  large 
number  of  these  organisms  are  robbed  of  their  vitality  by 
a  relatively  short  exposure  to  the  direct  rays  of  the  sun;  and 
it  is  therefore  not  unlikely  that  the  non-observance  of  this 
fact  may  be,  in  part  at  least,  accountable  for  some  of  the 
discrepancies  that  appear  in  the  results  of  these  experiments. 

In  his  studies  upon  the  behavior  of  pathogenic  and  other 
microorganisms  in  the  soil  Carl  Frankel1  found  that  micro- 
spira  comma  was  not  markedly  susceptible  to  those  dele- 
terious influences  that  cause  the  death  of  a  number  of  other 
pathogenic  organisms.  At  a  depth  of  one  and  a  half  meters 
vitality  was  not  destroyed,  and  there  was  a  regular  develop- 
ment in  cultures  so  placed. 

As  a  result  of  experiments  performed  in  the  Imperial 
Health  Bureau  at  Berlin,  it  was  found  that  the  bodies  of 
guinea-pigs  that  had  died  of  cholera  induced  by  Koch's 
method  of  inoculation  contained  no  living  cjiolera  spirilla 
when  exhumed  after  having  been  buried  for  nineteen  days 
in  wooden  boxes,  or  for  twelve  days  in  zinc  boxes.  In  a 

1  Zeitschrift  fur  Hygiene,  Bd.  ii,  S.  521. 


566      APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

few  that  had  been  buried  in  moist  earth,  without  having 
been  encased  in  boxes,  when  exhumed  after  two  or  three 
months,  the  results  of  examinations  for  cholera  spirilla  were 
likewise  negative. 

Esmarch1  found  that  when  the  cadaver  of  a  guinea-pig 
dead  after  the  introduction  of  cholera  organisms  into  the 
stomach  was  immersed  in  water  until  decomposition  was 
far  advanced,  it  was  impossible  to  find  any  living  microspira 
comma  by  the  ordinary  plate  methods.  Several  experi- 
ments resulted  in  their  disappearance  in  five  days.  In  one 
experiment,  in  which  decomposition  was  allowed  to  go  on 
without  the  animal  being  immersed  in  water,  none  could  be 
detected  after  the  fifth  day. 

Kitasato2  found  that  when  mixed  with  the  normal  intes- 
tinal evacuations  of  human  beings  it  lost  its  vitality  in  from 
a  day  and  a  half  to  three  days.  If  the  evacuations  were 
sterilized  before  the  cultures  were  mixed  with  them  it 
retained  its  vitality  from  twenty  to  twenty-five  days. 

Hesse3  and  Celli4  demonstrated  that  many  substances 
commonly  employed  as  food  serve  as  favorable  materials 
for  the  development  of  the  cholera  organisms. 

Kitasato5  found  that  at  36°  C.  microspira  comma  devel- 
oped very  rapidly  in  milk  during  the  first  three  or  four 
hours,  and  outnumbered  the  other  organisms  commonly 
present.  It  then  diminished  in  number  from  hour  to  hour 
as  the  acidity  of  the  milk  increased,  until  finally  its  vitality 
was  lost;  at  the  same  time  the  common  saprophytic  bac- 
teria increased  in  number.  Relatively  the  same  process 

1  v.  Esmarch,  Zeitschrift  fur  Hygiene,  Bd.  vii,  S.   1. 

2  Zeitschrift  fur  Hygiene,  Bd.  v,  S.  487. 

3  Ibid.,  S.  527. 

*  Bolletino  della  R.  Acad.  Med.  di.  Roma,  1888. 
&  Zeitschrift  fur  Hygiene,  Bd.  v,  S.  491. 


MICROSPIRA   COMMA  567 

occurs  at  a  lower  temperature,  from  22°  to  25°  C.;  but  it 
is  slower,  the  maximum  development  of  the  cholera  organ- 
isms being  reached  at  about  the  fifteenth  hour,  after  which 
time  they  were  outnumbered  by  the  ordinary  saprophytes 
present. 

From  the  foregoing  it  would  seem  that  the  vitality  of 
microspira  comma  in  milk  depends  largely  upon  the  reac- 
tion; the  more  quickly  the  milk  becomes-  sour  the  more 
quickly  does  the  organism  become  inert. 

According  to  Laser,1  the  cholera  organism  retains  its 
vitality  in  butter  for  about  seven  days;  it  is  therefore 
possible  for  the  disease  to  be  contracted  by  the  use  of  butter 
that  has  in  any  way  been  in  contact  with  cholera  material. 

When  dried  microspira  comma  retains  its  vitality  for 
from  about  three  to  twenty-four  hours,  according  to  the 
degree  of  desiccation.  In  moist  conditions  vitality  may  be 
retained  for  many  months;  though  repeated  observations 
lead  us  to  believe  that  under  these  circumstances  virulence 
is  diminished. 

Carbon  dioxide,  carbon  monoxide  and  nitrous  oxide  gases 
kill  this  germ  in  from  seven  to  ten  days. 

From  what  has  been  said,  we  see  that  the  spirillum  of 
Asiatic  cholera,  while  possessing  the  power  of  producing 
in  human  beings  one  of  the  most  rapidly  fatal  diseases  with 
which  we  are  acquainted,  is  still  one  of  the  least  resistant 
of  the  pathogenic  organisms  known  to  us.  Under  conditions 
most  favorable  to  its  growth  its  development  is  self -limited ; 
it  is  markedly  susceptible  to  acids,  alkalies,  other  chemical 
disinfectants,  and  heat;  but  when  partly  dried  upon  cloth- 
ing, food,  or  other  objects,  it  may  retain  its  vitality  for  a 
relatively  long  period  of  time,  and  it  is  more  than  probable 

1  Zeitachrift  fur  Hygiene,  Bd.  x,  S.  513. 


568     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

that  in  this  way  the  disease  is  often  disseminated  from  points 
in  which  it  is  epidemic  or  endemic  into  localities  that  are 
free  from  it. 

THE  DIAGNOSIS  OF  ASIATIC  CHOLERA  BY  BACTERIO- 
LOGICAL  METHODS. 

Because  of  the  manifold  channels  that  are  open  for  the 
ready  dissemination  of  this  disease  it  is  of  the  utmost  impor- 
tance that  it  should  be  recognized  as  quickly  as  possible,  for 
with  every  moment  of  delay  opportunities  for  its  spread 
multiply.  It  is  essential,  therefore,  when  employing  bac- 
teriological means  for  making  the  diagnosis,  to  bear  in  mind 
those  biological  and  morphological  features  of  the  organism 
that  appear  most  quickly  under  artificial  methods  of  cul- 
tivation, and  which,  at  the  same  time,  may  be  considered 
as  characteristic  of  it,  viz.,  its  peculiar  morphology  and 
grouping;  the  much  greater  rapidity  of  its  growth  over  that 
of  other  bacteria  with  which  it  may  be  associated;  the 
characteristic  appearance  of  its  colonies  on  gelatin  plates 
and  of  its  growth  in  stab-cultures  in  gelatin;  its  property 
of  producing  indol  and  coincidently  nitrites  in  from  six  to 
eight  hours  in  peptone  solution  at  37°  to  38°  C.;  and  its 
power  of  causing  the  death  of  guinea-pigs  in  from  sixteen 
to  twenty-four  hours  when  introduced  into  the  peritoneal 
cavity,  death  being  preceded  by  symptoms  of  extreme 
toxemia,  characterized  by  prostration  and  gradual  and 
continuous  fall  in  the  temperature  of  the  animal's  body. 

Koch1  devised  a  plan  of  procedure  that  comprehends  the 
points  just  enumerated.  By  its  employment  the  diagnosis 
can  be  established  in  the  majority  of  cases  of  Asiatic  cholera 

1  Zeitschrift  fur  Hygiene  und  Infektionskrankheiten,  1893,  Bd.  xiv,  S.  319. 


THE  DIAGNOSIS  OF  ASIATIC  CHOLERA  569 

in  from  eighteen  to  twenty-two  hours.  In  general,  the 
steps  to  be  taken  and  points  to  be  borne  in  mind  are  as 
follows: 

1.  Microscopic  Examination. — From  one  of  the  small  slimy 
particles  seen  in  the  semi-fluid  evacuations,   obtained  as 
soon  as  possible  after  their  passage,  prepare  a  cover-slip 
preparation  in  the  ordinary  way  and  stain  it.     If,  upon 
microscopic  examination,  only  curved  rods,  or  curved  rods 
greatly  in  excess  of  all  other  forms,  are  present,  the  diagnosis 
of  Asiatic  cholera  is  more  than  likely  correct;  and  particularly 
is  this  true  if  these  organisms  are  arranged  in  irregular  linear 
groups  with  the  long  axes  of  all  the  rods  pointing  in  nearly 
the  same  direction. 

2.  Plate  Cultures. — From  another  slimy  flake  prepare  a  set 
of  gelatin  plates.     Keep  them  at  a  temperature  of  from 
20°  to  22°  C.,  and  after  sixteen,  twenty-two,  and  thirty-six 
hours  observe  the  appearance  of  the  colonies.    Usually  after 
about  twenty-two  hours  the  colonies  of  this  organism  can 
easily  be  identified  by  one  familiar  with  them. 

3.  Peptone  Cultures. — With  another  slimy  flake  start  a 
culture  in  a  tube  of  peptone  solution — either  the  solution  of 
Dunham  or,  as  Koch  proposes,  a  solution  of  double  the 
strength  of  that  of  Dunham  (Witte's  peptone  is  to  be  used, 
as  it  gives  the  best  and  most  constant  results).    Keep  this 
at  from  37°  to  38°  C.,  and  at  the  end  of  from  six  to  eight 
hours  prepare  cover-slips  from  the  upper  layers  (without 
shaking)   and  examine  them  microscopically.     If  comma 
bacilli  were  present  in  the  original  material,  and  are  capable 
of  multiplication,  they  will  be  found  in  this  locality  in  almost 
pure  culture.     After  the  microscopic  examination  prepare 
a  second  peptone  culture  from  the  upper  layers  of  the  one 
just  examined,  also  a  set  of  gelatin  plates,  and  with  what 


570     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

remains  make  the  test  for  indol  by  the  addition  of  10  drops 
of  concentrated  sulphuric  acid  for  each  10  c.c.  of  fluid  con- 
tained in  the  tube.  If  comma  bacilli  are  growing  in  the  tube, 
the  rose  color  characteristic  of  the  presence  of  indol  should 
appear. 

By  following  this  plan  "a  bacteriologist  who  is  familiar 
with  the  morphological  and  biological  peculiarities  of  this 
organism  should  make  a  more  than  probable  diagnosis  at 
once  by  microscopic  examination  alone,  and  a  positive  diag- 
nosis in  from  twenty  to,  at  most,  twenty-four  hours  after 
beginning  the  examination."  (Koch.) 

Since  the  publication  of  the  foregoing  plan  many  other 
methods  have  been  suggested.  They  all  comprehend  the 
"enrichment,"  by  special  culture  methods,  of  the  number 
of  cholera  organisms  in  the  original  material  without  at 
the  same  time  encouraging  the  multiplication  of  the  other 
bacteria  present,  and  the  subsequent  isolation  of  the  cholera 
organism  by  the  use  of  selective  plating  media.  Of  these 
methods,  the  following  gives  general  satisfaction  and  can  be 
recommended : 

1.  Enrichment  in  the  peptone  solution  exactly  as  recom- 
mended above  by  Koch  if  it  be  intestinal  contents  that  are 
under  consideration;  if  it  be  water  or  sewage,  then  add  to 
90  c.c.  of  the  water  or  sewage  in  an  Erlinger  flask  10  c.c. 
of  a  10  per  cent,  solution.    Keep  at  37°  to  38°  C.  for  about 
eight  hours. 

2.  Without  shaking  the  tube  or  flask,  now  transfer  one 
wire  loopful  from  the  surface  of  the  mixture  of  feces,  water 
or  sewage  and  peptone  solution,  to  several  tubes  containing 
the  Benedict1  medium : 

Water 1000  c.c. 

Peptone 10  c.c. 

Sodium  chloride 5  c.c. 

1  Cent,  of  Bact.,  etc.,  Abt.  i,  Bd.  Ixii,  S.  536. 


THE  DIAGNOSIS  OF  ASIATIC  CHOLERA  571 

Boil  and  render  neutral  to  phenolphthalein. 
Add  1  grand  of  anhydrous  sodium  carbonate;  boil  and 
filter  through  double  filter  paper.    Add : 

Saccharose 5  grams 

Phenolphthalein  (sat.  sol.  in  50  per  cent,  alcohol)          5  c.c. 

Tube  and  sterilize  by  steam  at  100°  C. 

The  phenolphthalein  in  this  alkaline  solution  gives  to  the 
tubes  a  bright,  rose-red  color. 

As  the  vibrios  ferment  saccharose  rapidly,  with  resultant 
acid  production,  the  tubes  containing  them  are  quickly 
decolorized.  One,  therefore,  discards  all  tubes  that  are 
not  decolorized  after  eight  hours  at  37°  C.  Those  that  are 
decolorized  may  contain  cholera  vibrios  or  other  closely 
allied  spirilla  or  any  of  the  group  of  bacteria  having  the 
power  to  ferment  saccharose.  The  isolation  of  the  cholera 
spirilla  from  this  possible  mixture  is  now  accomplished  by 
differential  or  selective  plating. 

3.  Of  the  many  differential  plating  media  recommended, 
that  which  gives  uniformily  satisfactory  results  is  the 
alkaline  egg-medium  recommended  by  Krumwiede,  Pratt 
and  Grund,1  and  slightly  modified  by  Goldberg:2 

(a)  Alkaline  Egg  Solution. — Beat  up  a  whole  egg  with  an 
equal  volume  of  distilled  water.  Mix  an  equal  volume  of 
this  with  an  equal  volume  of  6.5  per  cent,  solution  of  anhy- 
drous sodium  carbonate  and  steam  for  from  one-half  to  one 
hour. 

(6)  Meat  extract-glucose-agar — 

Distilled  water 1000  c.c. 

Meat  extract  (Liebig)    . 3  grams 

Peptone  (Witte) 10  grams 

Sodium  chloride  (c.  p.) 5  grams 

Glucose 1  gram 

Agar-agar 30  grams 

1  Jour.  Infect.  Dis.,  1912,  x,  134. 

2  U.  S.  Pub.  Health  Service,  Hygiene  Lab.  Bull.,  1913,  No.  91,  p.  19. 


572     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

Steam  at  100°  C.,  for  three  hours  to  insure  complete  solution 
of  the  agar-agar. 

Decant,  or  filter  through  cotton,  and  distribute  in  100 
c.c.  flasks. 

Sterilize  by  steam  at  100°  C.  for  an  hour  and  a  half. 

For  use  mix  one  volume  of  a  with  5  volumes  of  6,  the 
latter  having  been  completely  liquefied  by  steam.  When 
thoroughly  mixed  pour  into  Petri  dishes,  to  a  depth  of  about 
3  mm.  in  each  dish,  and  allow  to  solidify.  When  the  medium 
is  solid,  the  dishes  may  be  placed  in  the  incubator  with  the 
covers  partly  removed  until  the  condensed  vapor  has  eva- 
porated. The  medium  should  be  comparatively  dry  before 
attempting  to  use  it.  When  dry  the  plates  so  prepared  may 
be  stored  in  dust  proof  receptacles  at  15°  C.  The  plates  may 
now  be  inoculated  from  the  surface  of  the  Benedict  medium. 
This  is  best  done  by  transferring  a  loopful  of  the  Benedict 
culture  to  the  surface  of  the  solid  alkaline  egg-glucose- 
agar  and  distributing  it  over  the  surface  with  a  sterile,  bent- 
glass  spreader.  When  thus  inoculated  the  plates  are  placed 
in  the  incubator  at  37°-38°  C.  until  colonies  develop. 

On  this  medium  the  cholera,  and  the  cholera-like  spirilla 
grow  luxuriantly,  while  the  other  bacteria  are  to  some  extent 
restrained. 

The  colonies  of  the  cholera  vibrios,  and  of  those  other 
vibrios  that  closely  resemble  it,  when  well  developed  on 
this  medium,  i.  e.,  after  about  twenty  hours  at  37°^38°  C., 
are,  to  the  naked  eye,  more  opaque  than  those  of  other 
bacteria;  under  the  low  power  lens  they  seem  as  if  in  the 
depths  of  the  medium,  are  more  or  less  hazy,  are  surrounded 
by  an  indistinct  halo  or  fringe  which  may  be  in  turn 
surrounded  by  a  clear  zone.  All  such  colonies  should  be 
examined  microscopically  and  from  all  that  are  composed 


THE  DIAGNOSIS  OF  ASIATIC  CHOLERA  573 

of  curved  or  spiral  organisms  pure  cultures  should  be  made 
for  subsequent  identification. 

In  abortive  cases  of  cholera  the  organisms  may  be  present 
in  the  intestinal  canal  in  very  small  numbers,  and  micro- 
scopic examination  is  not,  therefore,  of  so  much  assistance. 
In  these  cases  the  adoption  of  one  or  the  other  of  the  fore- 
going methods  is  imperative. 

In  the  foregoing  suggested  plans  it  will  be  observed  that 
plates  are  not  made  in  the  usual  way.  The  reason  for  this 
is  the  cholera  spirillum  being  markedly  aerobic  develops 
much  more  readily  on  the  surface  than  in  the  depths  of  the 
medium.  For  the  same  reason  the  material  taken  for 
plating  from  the  enriching  media  should  always  be  from 
the  surfaces,  without  the  tubes  or  flasks  having  been  shaken. 

It  being  desirable  to  have  the  colonies  isolated  from  one 
another  the  plates  should  be  relatively  dry,  that  is,  there 
should  be  no  collection  of  moisture  on  their  surfaces  that 
would  cause  the  colonies  to  become  confluent.  After  pour- 
ing, the  plates  should  always  be  kept  in  a  dust-free  incubator 
with  their  lids  off  until  all  excess  of  moisture  is  evaporated. 
All  colonies  of  curved  rods  should  be  isolated  in  pure  culture 
in  peptone  solution,  and  after  twenty  to  twenty-four  hours 
at  37°  to  38°  C.  such  cultures  should  be  tested  for  the 
presence  of  indol.  After  giving  positive  indol  reaction 
should  be  regarded  as  probably  cholera  spirilla. 

In  all  doubtful  cases,  in  which  only  a  few  curved  bacilli 
are  present,  or  in  which  irregularities  in  either  the  rate  or 
mode  of  their  development  occur,  pure  cultures  should  be 
obtained  as  soon  as  possible  and  their  virulence  tested  upon 
animals.  For  this  purpose  cultures  upon  agar-agar  from 
single  colonies  must  be  made.  From  the  surface  of  one  of 
such  cultures  a  large  wire-loopful  should  be  scraped  and 


574     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

broken  up  in  about  one  cubic  centimeter  of  physiological 
salt  solution,  and  the  suspension  thus  made  injected  by 
means  of  a  hypodermic  syringe  directly  into  the  peritoneal 
cavity  of  a  guinea-pig  of  about  350  to  400  grams  weight. 
For  larger  animals  more  material  is  used.  If  the  material 
injected  is  from  a  fresh  culture  of  the  cholera  organism,  toxic 
symptoms  at  once  appear;  these  have  their  most  pronounced 
expression  in  depression  of  temperature,  and  if  one  follows 
this  decline  in  temperature  from  time  to  time  with  the 
thermometer  it  will  be  seen  to  be  gradual  and  continuous 
from  the  time  of  injection  to  the  death  of  the  animal 
(Pfeiffer1),  which  occurs  in  from  eighteen  to  twenty-four 
hours  after  the  operation. 

MICROSPIRA  METCHNIKOVI  (GAMALEIA),  MIGULA,  1900. 

SYNONYM:  Vibrio  Metchnikovi.  Gamalela,  1888. 

A  spirillum  that  simulates  very  closely  the  comma  bacillus 
of  cholera  in  its  morphological  and  cultural  peculiarities, 
but  which  is  still  easily  distinguished  from  it,  is  that  de- 
scribed by  Gamalei'a2  under  the  name  of  microspira  Metch- 
nikovi. It  was  found  postmortem  in  a  number  of  fowls  that 
had  died  in  the  poultry-market  of  Odessa,  and  the  experi- 
ments of  the  discoverer  led  him  to  believe  that  it  was 
related  etiologically  to  the  gastro-enteritis  from  which  the 
chickens  had  been  suffering. 

Morphologically  it  appears  as  short,  curved  rods  and  as 
longer,  spiral-like  filaments.  It  is  usually  thicker  than 
Koch's  microspira  and  is  at  times  much  longer,  while  again 
it  is  seen  to  be  shorter.  It  is  usually  more  distinctly  curved 
than  the  "  comma  bacillus."  (Fig.  92.) 

1  Loc.  cit.      2  Annales  de  1'Institut  Pasteur,  1888,  tome  ii,  pp.  482,  552. 


MICROSPIRA   METCHNIKOVI  575 

It  is  supplied  with  a  single  flagellum  at  one  of  its  extremi- 
ties, and  is  therefore  motile. 
It  does  not  form  spores. 
It  is  aerobic. 

FIG.  92 


"        .'>• 

Microspira  Metchnikovi  from  agar-agar  culture,  twenty-four  hours  old. 

Its  growth  upon  gelatin  plates  is  usually  characterized, 
according  to  Pfeiffer,  by  the  appearance  of  two  kinds  of 
liquefying  colonies,  one  strikingly  like  those  of  the  Finkler- 
Prior  organism,  the  other  very  similar  to  those  produced 
by  Koch's  comma  bacillus,  though  in  both  cases  the  lique- 

FIG.  93 


Colony  of  microspira  Metchnikovi  in  gelatin,  after  thirty  hours  at  20°  to 
22°  C.     X  about  75  diameters. 


faction  resulting  from  the  growth  of  this  organism  is  more 
energetic  than  that  common  to  the  spirillum  of  Asiatic 
cholera.  After  from  twenty-four  to  thirty  hours  the  medium- 
size  colonies,  when  examined  under  a  low  power  of  the 


576     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

microscope,  show  a  yellowish-brown,  ragged  central  mass 
surrounded  by  a  zone  of  liquefaction  that  is  marked  by  a 
border  of  delicate  radii.     (Fig.  93.) 
In  gelatin  stab-cultures  the  growth  has  much  the  same 

FIG.  94 


abed 
Stab-culture  of  microspira  Metchnikovi  in  gelatin,  at  18°  to  20°  C.    a, 
after  twenty-four  hours;    6,  after  forty-eight  hours;    c,  after  seventy-two 
hours;  d,  after  ninety-six  hours. 


general  appearance  as  that  of  the  cholera  spirillum,  but  is 
exaggerated  in  degree.  The  liquefaction  is  far  more  rapid, 
and  the  characteristic  appearance  of  the  growth  is  lost  in 
from  three  to  four  days.  (See  a,  b,  c,  d,  Fig.  94.)  Develop- 
ment and  liquefaction  along  the  deeper  parts  of  the  needle- 


MICROSPIRA   METCHNIKOVI  577 

track  are  much  more  pronounced  than  is  the  case  with  the 
"comma  bacillus." 

Its  growth  on  agar-agar  is  rapid;  after  twenty-four  to 
forty-eight  hours  a  grayish  deposit  appears  which  has  a 
tendency  to  become  yellowish  with  age. 

On  potato  at  37°  C.  its  growth  is  seen  as  a  moist,  coffee- 
colored  patch,  surrounded  by  a  much  paler  zone.  The 
whole  growth  is  so  smooth  and  glistening  that  it  has  some- 
what the  appearance  of  being  varnished. 

In  bouillon  it  quickly  causes  opacity,  with  the  ultimate 
production  of  a  delicate  pellicle  upon  the  surface. 

It  causes  liquefaction  of  blood-serum,  the  liquefied  area 
being  covered  by  a  dense,  wrinkled  pellicle. 

When  grown  in  peptone  solution  it  produces  indol  and 
coincidently  nitrites,  so  that  the  rose-colored  reaction 
characteristic  of  indol  is  obtained  by  the  addition  of  sul- 
phuric acid  alone.  The  production  of  indol  by  this  organism 
is  usually  greater  than  that  common  to  the  comma  bacillus 
under  the  same  circumstances. 

In  milk  it  causes  an  acid  reaction  with  coagulation  of  the 
casein.  The  coagulated  casein  collects  at  the  bottom  of  the 
tube  in  irregular  masses,  above  which  is  a  layer  of  clear 
whey.  If  blue  litmus  has  been  added  to  the  milk,  the  color 
is  changed  to  pink  in  from  twenty-four  to  thirty  hours, 
and  after  forty-eight  hours  decolorization  and  coagulation 
occur.  The  clots  of  casein  are  not  re-dissolved.  After  about 
a  week  the  acidity  of  the  milk  is  at  its  maximum,  and  the 
organisms  quickly  die. 

It  causes  the  red  color  of  the  rosolic-acid-peptone  solution 
to  become  very  much  deeper  after  four  or  five  days  at  37°  C. 

It  does  not  cause  fermentation  of  glucose  with  production 
of  gas. 
37 


578      APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

It  is  killed  in  five  minutes  by  a  temperature  of  50°  C. 
(Sternberg.) 

It  is  pathogenic  for  chickens,  pigeons,  and  guinea-pigs. 
Rabbits  and  mice  are  affected  only  by  very  large  doses. 

Gamaleia  states  that  chickens  affected  with  the  choleraic 
gastro-enteritis  of  which  this  organism  is  the  cause,  are 
usually  seen  sitting  quietly  with  ruffled  feathers.  They 
suffer  from  diarrhea,  but  there  is  no  elevation  of  tempera- 
ture. Hyperemia  of  the  entire  gastro-intestinal  tract  is 
seen  at  autopsy.  The  other  internal  organs  do  not,  as  a 
rule,  present  anything  abnormal  to  the  naked  eye.  The 
intestinal  canal  contains  yellowish  fluid  with  which  blood 
may  be  mixed.  In  adult  chickens  the  spirilla  are  not  found 
in  the  blood,  but  in  young  ones  they  are  usually  present  in 
small  numbers. 

After  the  introduction  of  a  very  small  quantity  of  a  culture 
of  this  organism  directly  into  the  pectoral  muscle  pigeons 
succumb  in  from  eight  to  twenty  hours.  The  most  con- 
spicuous postmortem  lesion  is  found  at  the  site  of  inocula- 
tion. The  muscle  is  marked  by  yellow,  necrotic  stripes; 
is  more  or  less  edematous;  is  swollen,  and  contains  the 
vibrios  in  enormous  numbers.  The  intestines  are  usually 
filled  with  fluid  contents,  which  may  or  may  not  be  blood- 
stained; the  walls  of  the  intestines  are  often  injected  with 
blood,  and  occasionally  markedly  so.  The  conditions  of 
the  other  internal  viscera  are  inconstant.  In  fatal  cases 
the  vibrios  are  present  in  large  numbers  in  the  blood  and 
internal  organs.  In  pigeons'  that  survive  inoculation  the 
organisms  may  be  found  only  at  the  site  of  inoculation,  or 
very  sparingly  in  the  blood  also.  These  animals  usually 
exhibit  immunity  from  subsequent  inoculations.  In  certain 
instances  the  results  of -infection  are  chronic;  the  inoculated 


MICROSPIRA   METCHNIKOVI  579 

pectoral  muscle  atrophies,  the  pigeon  loses  in  weight  and 
finally  dies  after  one  or  two  weeks.  In  these  cases  the 
organisms  are  usually  absent  from  the  blood  and  internal 
organs,  and  may  even  be  absent  from  the  site  of  inoculation, 
or,  if  present,  in  only  very  small  number. 

Guinea-pigs  usually  die  in  from  twenty  to  twenty-four 
hours  after  subcutaneous  inoculation.  At  autopsy  an 
extensive  edema  of  the  subcutaneous  tissues  about  the  seat 
of  inoculation  is  seen,  and  there  is  usually  a  necrotic  condi- 
tion of  the  tissues  in  the  vicinity  of  the  point  of  puncture. 
As  the  blood  and  internal  organs  of  both  pigeons  and  guinea- 
pigs  contain  the  vibrios  in  large  numbers,  the  infection  in 
these  animals  takes,  therefore,  the  form  of  acute,  general 
septicemia. 

The  blood-serum  of  both  pigeons  and  guinea-pigs  that 
have  survived  inoculation  with  this  organism — i.  e.,  that 
have  acquired  immunity  from  it — is  bactericidal  in  vitro 
for  this  organism.  It  also  possesses  a  certain  degree  of 
immunity-conferring  property,  as  may  be  demonstrated  by 
injecting  it  into  normal  pigeons  and  guinea-pigs  that  are 
subsequently  to  be  inoculated  with  virulent  cultures. 

Very  old  cultures  of  this  organism  in  bouillon  become 
distinctly  alkaline  in  reaction.  At  this  stage  they  contain 
a  toxin  that  is  markedly  active  for  susceptible  animals. 
This  toxin  is  not  dissolved  in  the  fluid  to  any  extent,  but 
is  apparently  in  intimate  association  with  the  proteid  mat- 
ters composing  the  bacteria. 

Gastro-enteritis  may  be  produced  in  both  chickens  and 
guinea-pigs  by  feeding  them  with  food  with  which  cultures 
of  this  organism  have  been  mixed.  (Gamalei'a.) 


580     APPLICATION  OF  METHODS  OF   BACTERIOLOGY 
MICROSPIRA   SCHUYLKILLIENSIS,   ABBOTT,    1896. 

SYNONYM:    Vibrio  Schuylkilliensis,  Abbott,  1896. 

Abbott1  discovered  a  microspira  in  the  water  of  the 
Schuylkill  River,  at  Philadelphia,  and  later,  Bergey2  reports 
the  presence  of  the  same  organism,  as  well  as  several  varieties 
that  are  slightly  different,  in  the  waters  of  the  Schuylkill 
and  Delaware  rivers,  along  the  entire  city  front,  more 
especially  in  the  effluents  of  the  sewers. 

Microspira  Schuylkilliensis  is  a  short,  rather  plump 
"comma,"  often  with  a  very  decided  curve,  with  rounded 
or  slightly  pointed  ends.  As  usually  seen  it  is  a  little  shorter 
and  thicker  than  the  microspira  comma,  though  this  feature 
is  quite  variable.  It  is  actively  motile,  having  a  single  polar 
flagellum.  It  does  not  form  spores.  It  stains  with  the 
ordinary  aniline  stains,  but  is  negative  to  Gram's  method. 

The  colonies  on  gelatin  are  sharply  defined,  distinctly 
granular,  and  have  usually  fine  irregular  markings,  as  if 
they  were  creased  or  folded.  Sometimes  they  present 
indistinct  concentric  markings.  As  growth  progresses  these 
markings  become  more  and  more  distinct  and  finally  give 
to  the  colony  a  decidedly  lobulated  or  mulberry-like  ap- 
pearance. 

After  about  the  third  or  fourth  day,  when  liquefaction  is 
actively  in  progress,  the  majority  of  the  colonies  lose  their 
characteristic  appearance.  They  are  seen  as  irregular, 
ragged,  granular  masses  lying  in  the  center  of  pits  of  lique- 
fied gelatin. 

In  stab  cultures  in  gelatin  the  appearance  of  the  growth  is 
essentially  that  of  microspira  comma,  though  at  times  it  is 
a  little  more  rapid  in  progress. 

1  Jour,  of  Exp.  Med.,  1896,  i,  419.  2  Ibid.,  1897,  ii,  535. 


MICROSPIRA  SCHUYLKILLIENSIS  581 

On  meat-infusion  agar-agar,  neutral  or  slightly  alkaline 
to  phenolphthalein,  growth  is  very  rapid  at  the  body  tem- 
perature. The  general  character  of  the  growth  corresponds 
to  that  of  microspira  comma. 

The  growth  on  blood  serum,  after  twenty-four  hours-  at 
body  temperature,  appears  as  a  line  of  depression,  which 
increases  as  a  track  of  liquefaction,  and  later  results  in  the 
more  or  less  complete  liquefaction  of  the  medium. 

Bouillon  becomes  uniformly  clouded  in  twenty-four  hours 
at  the  body  temperature.  Its  reaction  becomes  more  alkaline 
as  growth  progresses.  A  pellicle,  at  first  delicate,  later 
denser,  always  characterizes  the  growth  in  this  medium. 

Usually  no  visible  growth  occurs  on  a  potato. 

In  fresh  litmus-milk  a  slight  degree  of  acidity  is  noticed 
after  twenty-four  hours  at  body  temperature.  After  forty- 
eight  hours  this  acidity  is  slightly  greater,  and  at  tunes  the 
milk  shows  evidences  of  coagulation,  though  not  always. 

Microspira  Schuylkilliensis  is  a  facultative  aerobe.  In 
fluid  media  under  an  atmosphere  of  carbon  dioxide  in  sealed 
tubes  no  growth  is  observed. 

The  organism  grows  most  luxuriantly  at  about  37.5° 
C.  Growth  is  hardly  perceptible  at  10°  C.  It  is  destroyed 
by  an  exposure  of  five  minutes  to  50°  C. 

None  of  the  carbohydrates  are  broken  up  with  the  libera- 
tion of  gas. 

It  produces  indol  and  at  the  same  time  reduces  nitrates 
to  nitrites. 

The  pathogenic  properties  of  this  organism  are  best  seen 
in  guinea-pigs  and  pigeons,  both  of  which  are  uniformly 
susceptible.  Rabbits  and  chickens  resist  relatively  large 
doses.  Mice  are  infected  with  small  doses  injected 
subcutaneously. 


582     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

The  most  characteristic  lesions  follow  the  injection  of 
cultures  into  the  pectoral  muscles  of  pigeons.  At  death 
the  inoculated  muscle  is  swollen,  necrotic,  and  the  overlying 
tissues  are  edematous.  The  bacteria  are  found  in  large 
numbers  in  the  vicinity  of  the  seat  of  the  inoculation, 
and  in  relatively  small  numbers  in  the  blood  and  internal 
organs. 


CHAPTER  XXVII. 

Study  of  Bacterium  Anthracis,  and  of  the  Effects  Produced  by  Its  Inocu- 
lation into  Animals — Peculiarities  of  the  Organism  Under  Varying  Con- 
ditions of  Surroundings — Anthrax  Vaccines — Anthrax  Immune  Serum. 

THE  discovery  that  the  blood  of  animals  suffering  from 
splenic  fever,  or  anthrax,  always  contains  minute  rod-shaped 
bodies  (Pollender,  1855;  Davaine,  1863),  led  to  a  group  of 
investigations  that  have  not  only  fully  familiarized  us  with 
the  nature  of  this  malady  in  particular,  but  have  perhaps 
contributed  more,  incidentally,  to  our  knowledge  of  bac- 
teriology in  general  than  studies  upon  any  other  single 
infective  process  or  its  causative  agent. 

The  direct  outcome  of  these  investigations  is  that  a  rod- 
shaped  microorganism,  now  known  as  bacterium  anthracis, 
is  always  present  in  the  blood  of  animals  suffering  from  this 
disease;  that  this  organism  can  be  obtained  from  the  tissue 
of  those  animals  in  pure  cultures;  and  that  such  artificial 
cultures  of  bacterium  anthracis  when  introduced  into  the 
bodies  of  susceptible  animals  can  again  produce  a  condition 
identical  with  that  found  in  the  animal  from  which  they 
were  obtained.  The  disease  is  a  true  septicemia,  and  after 
death  the  capillaries  throughout  the  body  are  always  found 
to  contain  the  typical  rod-shaped  organism  in  larger  or 
smaller  numbers. 

This  organism,  when  isolated  in  pure  culture,  is  a  bac- 
terium which  varies  considerably  in  length,  ranging  from 
short  rods,  2  to  3^  in  length,  to  longer  threads,  20  to  25/x 

(583) 


584      APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

in  length.  In  breadth  it  is  from  1  to  1.25/z.  Frequently 
very  long  threads,  made  up  of  several  rods  joined  end  to 
end,  are  seen. 

When  obtained  directly  from  the  body  of  an  animal  it 
is  usually  in  the  form  of  short  rods  square  at  the  ends.  If 
highly  magnified,  the  ends  are  seen  to  be  a  trifle  thicker 
than  the  body  of  the  cell  and  somewhat  indented  or  concave, 
suggestive  of  the  joints  of  bamboo,  peculiarities  that  help  to 
distinguish  it  from  certain  other  organisms  that  are  some- 
what like  it  morphologically.  (See  Fig.  95.) 

FIG.  95 


Bacterium  anthracis,  highly  magnified  to  show  swellings  and  concavities  at 
extremities  of  the  single  cells. 


When  cultivated  artificially  at  the  temperature  of  the 
body  the  bacterium  of  anthrax  presents  a  series  of  very 
interesting  developmental  phases. 

The  short  rods  grow  into  long  threads,  which  may  be 
seen  twisted  or  plaited  together  like  ropes,  each  thread  being 
marked  by  the  points  of  juncture  of  the  segments  com- 
posing it.  (Fig.  96,  a  and  6.)  In  this  condition  it  remains 
until  alterations  in  its  surroundings,  the  most  conspicuous 
being  diminution  of  its  nutritive  supply,  favor  the  produc- 
tion of  spores.  When  this  stage  begins  changes  in  the  proto- 
plasm may  be  noticed;  the  bacteria  become  marked  by 
irregular  granular  bodies,  which  eventually  coalesce  into 


BACTERIUM  ANTHRACIS  585 

glistening  oval  spores,  one  of  which  lies  in  nearly  every 
segment  of  the  long  thread,  and  gives  to  the  thread  the 
appearance  of  a  string  of  shining  beads.  (Fig.  97.)  In 

FIG.  96 

§"*S«£ 
f  «~^- 

i  *ft 


TV 

a  '»*  6 

Bacterium  anthracis.     Plainted  and  twisted  threads  seen  in  fresh-growing 
cultures.    .X  about  400  diameters. 

this  stage  they  remain  but  a  short  time.  The  chains  of 
spores,  which  are  held  together  by  the  remains  of  the  cells 
in  which  they  formed,  become  broken  up,  and  eventually 
nothing  but  free  oval  spores,  and  here  and  there  the  remains 

FIG.  97 


Threads  of  bacterium  anthracis  containing  spores.     X  about  1200  diameters. 

of  mature  bacilli  which  have  undergone  degenerative  changes, 
can  be  found.  In  this  condition  the  spores,  capable  of  resist- 
ing deleterious  influences,  remain  and,  unless  their  sur- 


586     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

roundings  are  altered,  continue  in  this  living,  though  inactive, 
condition  for  a  very  long  time.  If  again  placed  under  favor- 
able conditions,  each  spore  will  germinate  into  a  mature  cell, 
and  the  same  series  of  changes  will  be  repeated  until  the 
surroundings  become  again  gradually  unfavorable  to  develop- 
ment, when  spore-formation  again  takes  place.  Spore  forma- 
tion occurs  only  at  temperatures  ranging  from  18°  to  43°  C.; 
37.5°  C.  being  the  optimum.  Under  12°  C.  they  are  not 
formed.  (Why?)  This  organism  does  not  form  spores  in 
the  tissues  of  the  living  animal,  its  usual  condition  at  this 

FIG.  98 


Colony  of  bacterium  anthracis  on  agar-agar. 

time  being  that  of  short  rods;  occasionally,  however,  some- 
what longer  forms  may  be  seen. 

The  bacterium  of  anthrax  is  not  motile. 

Colonies  of  this  organism,  as  seen  upon  agar-agar,  present 
a  typical  appearance,  from  which  they  have  been  likened 
unto  the  head  of  Medusa.  From  a  central  point,  which  is 
more  or  less  dense/  consisting  of  a  felt-like  mass  of  long 
threads  irregularly  matted  together,  the  growth  continues 
outward  upon  the  surface  of  the  agar-agar  (Fig.  98.)  It 
is  made  up  of  wavy  bundles  in  which  the  threads  are  seen 
to  lie  parallel  or  are  twisted  in  strands  like  those  of  a  rope; 


BACTERIUM  ANTHRACIS  587 

sometimes  they  have  a  plaited  arrangement.  (See  Fig.  96.) 
These  bundles  twist  and  cross  in  all  directions,  and  even- 
tually disappear  at  the  periphery  of  the  colony.  At  the 
extreme  periphery  of  the  colonies  it  is  sometimes  possible 
to  trace  single  bundles  of  these  threads  for  long  distances 
across  the  surface  of  the  agar-agar.  The  colony  itself  is 
not  circumscribed  in  appearance,  but  is  more  or  less  irregu- 
larly fringed  or  ragged,  or  scalloped.  To  the  naked  eye 
they  look  very  much  like  minute  pellicles  of  raw  cotton  that 
have  been  pressed  into  the  surface  of  the  agar-agar. 

As  the  colonies  continue  to  grow  they  become  more  and 
more  dense  and  opaque,  and  granular  and  rough  on  the 
surface.  When  touched  with  a  sterilized  needle  one  experi- 
ences a  sensation  that  suggests  somewhat  their  matted 
structure.  They  are  never  moist  or  creamy.  The  bit  that 
is  taken  up  with  the  needle  is  always  more  or  less  ragged, 
suggesting  a  tiny  particle  of  moist  blotting  paper. 

The  colonies  on  gelatin  at  the  earliest  stages  also  present 
the  same  wavy  appearance;  but  this  characteristic  soon 
becomes  in  part  destroyed  by  the  liquefaction  of  the  gelatin 
which  is  produced  by  the  growing  organisms.  This  allows 
them  to  sink  to  the  bottom  of  the  fluid,  where  they  lie  as 
irregular  masses.  Through  the  fluid  portion  of  the  gelatin 
may  be  seen  small  clumps  of  growing  bacteria,  which  look 
very  much  like  bits  of  cotton-wool. 

In  bouillon  the  growth  is  characterized  by  the  formation 
of  flaky  masses,  which  also  have  very  much  the  appearance 
of  bits  of  raw  cotton.  Microscopic  examination  of  one  of 
these  flakes  reveals  the  twisted  and  plaited  arrangement  of 
the  long  threads. 

On  potato  it  develops  rapidly  as  a  dull,  dry,  granular, 
whitish  mass,  which  is  more  or  less  limited  to  the  point  of 


588     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

inoculation.  On  potato,  at  the  temperature  of  the  incubator, 
spore-formation  may  be  easily  observed. 

Stab-  and  slant-cultures  on  agar-agar  present  in  general 
the  appearances  given  for  the  colonies,  except  that  the 
growth  is  much  more  extensive.  The  growth  is  always 
more  pronounced  on  the  surface  than  down  the  track  of  the 
needle. 

On  gelatin  it  causes  liquefaction,  which  begins  on  the 
surface  at  the  point  inoculated  and  spreads  outward  and 
downward. 

It  grows  best  with  access  to  oxygen,  and  very  poorly  when 
the  supply  of  that  gas  is  interfered  with. 

Under  favorable  conditions  of  aeration,  nutrition,  and 
temperature  its  growth  is. rapid. 

Under  12°  C.  and  above  45°  C.  no  growth  occurs.  Its 
optimum  temperature  is  that  of  the  body,  viz.,  37°-38°  C. 

The  spores  of  bacterium  anthracis  are  very  resistant  to 
heat,  though  the  degree  of  resistance  varies  with  spores 
of  different  origin.  It  has  been  found  repeatedly  that 
anthrax  spores  from  some  strains  are  readily  killed  by  an 
exposure  of  one  minute  to  the  temperature  of  steam,  whereas 
spores  from  others  resist  this  temperature  for  as  long  as 
twenty  minutes  and  sometimes  longer. 

Staining. — Anthrax  bacteria  stain  readily  with  the 
ordinary  aniline  dyes.  In  tissues  their  presence  may  also 
be  demonstrated  by  the  ordinary  aniline  staining  fluid 
or  by  Gram's  method.  They  may  also  be  stained  in  tissues 
with  a  strong  watery  solution  of  dahlia,  after  which  the 
sections  are  decolorized  in  2  per  cent,  sodium  carbonate 
solution,  washed  in  water,  dehydrated  in  alcohol,  cleared 
in  xylol,  and  mounted  in  balsam.  This  leaves  the  bacilli 
stained,  while  the  tissues  containing  them  are  decolorized; 


BACTERIUM  ANTHRACIS  589 

or  the  latter  may  be  stained  a  contrast-color — with  eosin, 
for  example — after  dehydration  in  alcohol  and  before 
clearing  in  xylol.  In  this  case  they  must  be  washed  again 
in  alcohol  before  using  the  xylol.  In  a  preparation  treated 
in  this  way  the  rod-shaped  organisms  are  of  a  purple  color, 
ano^  will  be  seen  in  the  capillaries  of  the  tissues,  while  the 
tissues  themselves  are  of  a  pale  rose  color. 

Inoculation  into  Animals. — Introduce  into  the  subcutaneous 
tissues  of  the  abdominal  wall  of  a  guinea-pig  or  rabbit  a 
portion  of  a  pure  culture  of  bacterium  anthracis.  The  animal 
usually  succumbs  in  from  thirty-six  to  forty-eight  hours. 
Little  or  no  reaction  at  the  immediate  point  of  inoculation 
will  be  noticed;  but  beyond  this,  extending  for  a  long  dis- 
tance over  the  abdomen  and  thorax,  the  tissues  will  be 
markedly  edematous.  Here  and  there,  scattered  through 
this  edematous  tissue,  small  ecchymoses  will  be  seen.  The 
underlying  muscles  are  pale  in  color.  Inspection  of  the 
internal  viscera  reveals  no  very  marked  macroscopic  changes 
except  in  the  spleen.  This  is  enlarged,  dark  in  color,  and 
soft.  The  liver  may  present  the  appearance  of  cloudy 
swelling;  the  lungs  may  be  red  or  pale  red  in  color;  the 
heart  is  usually  filled  with  blood.  No  other  changes  can 
be  seen  by  the  naked  eye. 

Prepare  cover-slip  preparations  from  the  blood  and  other 
viscera.  They  will  all  be  found  to  contain  short  rods  in 
large  numbers.  Nowhere  can  spore-formation  be  detected. 
Upon  microscopic  examination  of  sections  of  the  organs 
which  have  been  hardened  in  alcohol  the  capillaries  are  seen 
to  be  filled  with  the  bacteria;  in  some  places  closely  packed 
in  large  numbers,  at  other  points  fewer  in  number.  Usually 
they  are  present  in  largest  numbers  in  those  tissues  having 
the  greatest  capillary  distribution  and  at  those  points  at 


590      APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

which  the  circulation  is  slowest.  They  are  uniformly  dis- 
tributed through  the  spleen.  The  glomeruli  of  the  kidneys 
and  the  capillaries  of  the  lungs  are  frequently  packed  with 
them.  The  capillaries  of  the  liver  contain  them  in  large 
numbers.  (Fig.  99.)  Hemorrhages,  probably  due  to 
rupture  of  capillaries  by  the  mechanical  pressure  of  the 
bacteria  which  are  developing  within  them,  not  uncommonly 
occur.  When  these  occur  in  the  mucous  membranes  of  the 
alimentary  tract  the  blood  may  escape  through  the  mouth 


Bacterium  anthracis  in  liver  of  mouse.     X  about  450  diameters.     Bacteria 
stained  by  Gram's  method;    tissue  stained  with  Bismarck-brown. 


or  anus;  when  in  the  kidneys,  through  the  uriniferous 
tubules. 

Cultures  from  the  different  organs  or  from  the  edematous 
fluid  about  the  point  of  inoculation  result  in  growth  of 
bacterium  anthracis. 

The  amphibia,  dogs,  and  the  majority  of  birds  are  not 
susceptible  to  this  disease.  Rats  are  difficult  to  infect. 
Rabbits,  guinea-pigs,  white  mice,  gray  house-mice,  sheep, 
and  cattle  are  susceptible.  Infection  may  occur  either 


BACTERIUM  ANTHRACIS  591 

through  the  circulation,  through  the  air-passages,  through 
the  alimentary  tract,  or,  as  we  have  just  seen,  through  the 
subcutaneous  tissues. 

Protective  Inoculation. — The  most  noteworthy  application 
of  artificially  prepared  living  vaccines  to  the  protection  of 
animals  from  infection  is,  seen  in  connection  with  anthrax 
in  sheep  and  in  bo  vines. 

By  a  variety  of  procedures  the  virulent  anthrax  bacterium 
may  be  in  part  or  totally  robbed  of  its  pathogenic  properties. 
It  is  through  the  very  mild  constitutional  disturbance 
caused  in  animals  vaccinated  with  such  weakened  cultures 
that  protection  is  often  afforded  against  the  severer,  fre- 
quently fatal,  form  of  the  infection. 

Without  reviewing  the  various  methods  that  have  been 
employed  for  attenuating  the  virulence  of  this  organism  to 
a  degree  suitable  for  protective  vaccination,  it  will  suffice 
to  say  that  the  most  satisfactory  results  have  been  obtained 
by  the  classical  method  of  Pasteur.  This  comprehends  the 
long-continued  cultivation,  (ten  to  thirty  days)  at  a  tem- 
perature of  from  42°  to  43°  C.  In  this  procedure  the  spore- 
free,  virulent  bacterium  anthracis,  obtained  directly  from 
the  blood  of  a  recently  dead  animal,  is  brought  at  once  into 
sterile  nutrient  bouillon  in  about  twenty  test-tubes,  which 
are  immediately  placed  in  an  incubator  that  is  carefully 
regulated  to  maintain  a  temperature  of  42.5°  C.  There 
should  not  be  a  fluctuation  of  over  0.1°  C. 

After  about  a  week  a  tube  is  removed  from  the  incubator 
on  each  successive  day  and  its  virulence  tested  at  once  on 
animals.  The  degree  of  attenuation  experienced  by  the 
cultures  grown  under  these  circumstances  is  determined  by 
tests  upon  rabbits,  guinea-pigs,  and  mice.  The  first  culture 
removed  may  or  may  not  kill  rabbits,  the  most  resistant 


592     APPLICATION  OF  METHODS  OF   BACTERIOLOGY 

of  the  three  animals  used  for  the  test,  while  it  will  certainly 
kill  the  guinea-pigs  and  mice;  after  another  two  or  three 
days  rabbits  will  no  longer  succumb  to  inoculation  with  the 
culture  last  removed  from  the  incubator,  while  no  diminu- 
tion will  as  yet  be  noticed  in  its  pathogenesis  for  the  other 
two  species.  After  four  to  seven  ^days  more  a  culture  may 
be  encountered  that  kills  only  mice,  the  guinea-pigs  escap- 
ing; while  ultimately,  if  the  experiment  be  continued,  a  degree 
of  attenuation  may  be  reached  in  which  the  organism  has 
not  even  the  power  of  killing  a  mouse,  though  it  still  retains 
its  vitality.  Investigation  of  these  attenuations  shows 
them  to  possess  all  the  characteristics  of  enfeebled  anthrax 
bacteria;  they  grow  slowly  and  less  vigorously  when  trans- 
planted; they  do  not  form  spores  when  exposed  to  a  high 
temperature;  and  microscopically  they  present  evidences 
of  degeneration.  When  introduced  beneath  the  skin  of 
animals  they  disseminate  but  slightly  beyond  the  site  of 
inoculation,  and  do  not,  as  a  rule,  cause  the  general  septicemia 
that  occurs  in  susceptible  animals  inoculated  with  normal 
cultures  of  this  organism.  In  the  practical  employment  of 
these  attenuated  cultures  for  protective  purposes  two 
vaccines  are  employed.  These  were  designated  by  Pasteur 
as  "first"  and  "second"  vaccines.  The  "first"  is  the  one 
that  killed  only  the  mice  in  the  preliminary  tests;  while 
the  "  second"  is  that  which  killed  both  mice  and  guinea-pigs, 
but  failed  to  kill  the  rabbit.  When  larger  animals,  such  as 
sheep  or  cattle,  are  to  be  protected  by  vaccination  with 
these  vaccines,  a  subcutaneous  inoculation  of  about  0.3  c.c. 
of  the  first  vaccine  is  usually  given.  This  should  be  prac- 
tically without  noticeable  effect,  causing  neither  rise  of 
body-temperature  nor  other  constitutional  or  local  symp- 
toms. After  a  period  of  about  two  weeks  the  second  vaccine 


BACTERIUM  ANTHRACIS  593 

is  injected  in  the  same  way;  this  may  or  may  not  cause 
disturbance.  In  the  event  of  its  doing  so  the  symptoms  are 
rarely  alarming,  and,  if  the  vaccines  have  been  properly 
prepared  and  tested  before  use,  all  symptoms  disappear 
within  a  short  time  after  the  injection. 

In  the  large  majority  of  cases  sheep,  bovines,  horses,  and 
mules  may  be  safely  protected  against  anthrax  by  the  careful 
practice  of  this  method. 

Sobernheim1  found  that  it  was  possible  to  bring  about  a 
high  degree  of  immunity  against  bacterium  anthracis  by 
means  of  the  vaccines  1  and  2  of  Pasteur,  with  subsequent 
inoculations  of  virulent  organisms.  He  employed  the  serum 
of  animals  thus  immunized  in  the  treatment  of  sheep  that 
had  been  injected  with  highly  virulent  anthrax  bacteria. 
Five  sheep  were  treated  in  this  way,  and  all  of  them  recovered 
with  only  slight  rise  in  temperature  and  moderate  infiltra- 
tion at  the  point  of  injection,  while  control  animals  died 
very  promptly. 

He  further2  reports  an  improvement  on  the  method  of 
protective  inoculation  against  anthrax  in  which  he  uses  a 
combination  of  anthrax  vaccines  and  immune  serum,  in 
which  the  results  are  far  more  satisfactory  than  with  the 
anthrax  vaccines  alone.  He  states  that  this  new  method 
has  the  following  advantages  over  the  Pasteur  method: 
(1)  That  the  immunization  can  be  carried  out  without 
losing  any  of  the  animals;  (2)  that  it  can  be  completed 
in  one  day;  (3)  that  stronger  and  more  active  cultures 
can  be  employed  and  therefore  a  more  durable  immunity 
obtained;  and  (4)  that  the  serum  alone  can  be  employed  as 
a  curative  agent. 

1  Berliner  klin.  Wchnschr.,  1897. 

2  Ibid.,  1902,  p.  516. 
38 


594     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

Anthrax  Immune  Serum. — Sanfelice1  experimented  with 
the  serum  of  dogs  that  had  been  immunized  from  anthrax 
bacteria.  This  serum  possessed  immunizing  and  curative 
properties,  as  shown  by  experiments  upon  animals.  He  had 
an  opportunity  of  trying  the  serum,  with  favorable  results, 
upon  a  man  who  had  contracted  anthrax.  The  total  amount 
of  serum  employed  was  56  cubic  centimeters.  There  was 
no  reaction  at  the  point  of  injection  of  the  serum.  The 
therapeutic  effect  of  the  administration  of  serum  was  a 
general  improvement  in  the  symptoms,  marked  fall  of  the 
temperature  on  the  second,  and  complete  apyrexia  on  the 
third  day.  The  effect  on  the  local  anthrax  lesion  manifested 
itself  in  reduction  and,  finally,  disappearance  of  the  edema, 
followed  first  by  an  increased  swelling  of  the  glands,  which 
decreased  again  subsequently.  He  states  that  the  serum 
treatment  should  be  continued  not  only  till  the  temperature 
has  fallen  to  normal  and  a  diminution  of  the  edema  is 
apparent,  but  also  until  there  is  marked  reduction  in  the 
size  of  the  swollen  lymph-glands. 

Sclavo2  immunized  a  number  of  animals,  principally 
sheep  and  goats,  with  the  two  vaccines  of  Pasteur,  followed 
by  repeated  injections  of  increasing  quantities  of  virulent 
cultures.  By  this  means  he  obtained  an  immune  serum 
which  had  protective  as  well  as  curative  properties  when 
tested  upon  guinea-pigs  and  rabbits. 

Cicognani3  employed  Sclavo's  immune  serum  on  12  per- 
sons suffering  from  various  grades  of  anthrax  infection, 
some  of  the  cases  being  severe  infections  in  which  the  prog- 
nosis would  otherwise  have  been  very  unfavorable.  The 

1  Centralblatt  f.  Bacteriologie,  Originate,   1902,  Bd.  xxxiii. 

2  Bulletin  de  1'Institut  Pasteur,  T.  I.,  1903,  p.  305.  . 

3  Centralblatt  f.  Bacteriologie,  1902,  ref.  Bd.  31,  p.  725, 


BACTERIUM  ANTHRACIS  595 

duration  of  the  disease  was  always  very  much  shortened 
and  all  recovered. 

Lazaretti1  reports  23  cases  of  human  infection  with  bac- 
terium anthracis  in  which  Sclavo's  immune  serum  was 
employed  with  recovery  in  each  case.  Another  patient, 
suffering  from  chronic  alcoholism  and  malaria,  did  not 
recover. 

Experiments. — Prepare  three  cultures  of  bacterium  an- 
thracis— one  upon  gelatin,  one  upon  agar-agar,  and  one  upon 
potato.  Allow  the  gelatin  culture  to  remain  at  the  ordinary 
temperature  of  the  room,  place  the  agar-agar  culture  in  the 
incubator,  and  the  potato  culture  at  a  temperature  not 
above  18°  to  20°  C.  Prepare  cover-slips  from  each  from 
day  to  day.  What  differences  are  observed? 

Prepare  two  potato  cultures  of  bacterium  anthracis. 
Place  one  in  the  incubator  and  maintain  the  other  at  a 
temperature  of  from  18°  to  20°  C.  Examine  them  each  day. 
Do  they  develop  in  the  same  way? 

From  a  fresh  culture  of  bacterium  anthracis,  in  which 
spore-formation  is  not  yet  begun  (which  is  the  surest  source 
from  which  to  obtain  non-spore-bearing  anthrax  bacteria?), 
prepare  a  hanging-drop  preparation;  also  a  cover-slip 
preparation  in  the  usual  way  and  stain  it  with  a  strong 
gentian- violet  solution;  and  another  cover-slip  preparation 
which  is  to  be  drawn  through  a  flame  twelve  to  fifteen  times, 
stained  with  aniline  gentian-violet,  washed  in  iodine  solu- 
tion and  then  in  water.  Examine  these  microscopically. 
Do  they  all  present  the  same  appearance?  To  what  are 
the  differences  due? 

1  Deutsche  Viertel j  ahrsschrif t  f.  offentliche  Gesundheitspflege,  1903, 
Bd.  xxxv,  Supplement,  p.  253. 


596     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

Do  the  anthrax  threads,  as  seen  in  a  fresh,  growing, 
hanging  drop,  present  the  same  morphological  appearance 
as  when  dried  and  stained  upon  a  cover-slip?  How  do  they 
differ? 

Liquefy  a  tube  of  agar-agar,  and  when  it  is  at  the  tem- 
perature of  40°  to  43°  C.  add  a  very  minute  quantity  of  an 
anthrax  culture  which  is  far  advanced  in  the  spore-stage. 
Mix  it  thoroughly  with  the  liquid  agar-agar  and  from  this 
prepare  several  hanging  drops  under  strict  antiseptic  pre- 
cautions, using  the  fluid  agar-agar  for  the  drops  instead 
of  bouillon  or  salt  solution.  Select  from  among  these 
preparations  that  one  in  which  the  smallest  number  of 
spores  are  present.  Under  the  microscope  observe  the 
development  of  a  spore  into  a  mature  cell.  Describe  care- 
fully the  developmental  stages. 

Prepare  a  1  :  1000  solution  of  carbolic  acid  in  bouillon. 
Inoculate  this  with  virulent  anthrax  spores.  If  no  develop- 
ment occurs  after  two  or  three  days  at  the  temperature  of 
the  thermostat,  prepare  a  solution  of  1  :  1200,  and  continue 
until  the  point  is  reached  at  which  the  amount  of  carbolic 
acid  present  jwt  permits  of  the  development  of  the  spores. 
When  the  proper  dilution  is  reached  prepare  a  dozen  of 
such  tubes  and  inoculate  one  of  them  with  virulent  anthrax 
spores.  As  soon  as  development  is  well  advanced  transfer 
a  loopful  from  this  tube  into  a  second  of  the  carbolic  acid 
tubes;  when  this  has  developed,  then  from  this  into  a  third, 
etc.  After  five  or  six  generations  have  been  treated  in  this 
way  study  the  spore  production  of  the  organisms  in  that 
tube.  If  it  is  normal,  continue  to  inoculate  from  one  car- 
bolic acid  tube  to  another,  and  see  if  it  is  possible  by  this 


BACTERIUM  ANTHRACIS  597 

means  to  influence  in  any  way  the  production  of  spores  by 
the  organism  with  which  you  are  working.  What  is  the 
effect,  if  any? 

Prepare  two  bouillon  cultures,  each  from  one  drop  of 
blood  of  an  animal  dead  of  anthrax.  (Why  from  the  blood 
of  an  animal  and  not  from  a  culture?)  Allow  one  of  them 
to  grow  for  from  fourteen  to  eighteen  hours  in  the  incubator; 
allow  the  other  to  grow  at  the  same  temperature  for  three 
or  four  days.  Remove  the  first  tube  after  the  time  men- 
tioned and  subject  it  to  a  temperature  of  80°  C.  for  thirty 
minutes.  At  the  end  of  this  time  prepare  four  plates  from 
it.  Make  each  plate  with  one  drop  from  the  heated  bouillon 
culture.  At  the  end  of  three  or  four  days  treat  the  second 
tube  in  identically  the  same  way.  How  do  the  number  of 
colonies  which  develop  from  the  two  cultures  compare? 
Was  there  any  difference  in  the  time  required  for  their 
development  on  the  plates? 

From  a  potato  culture  of  bacterium  anthracis  which  has 
been  in  the  incubator  for  three  or  four  days  scrape  away 
the  growth  and  carefully  break  it  up  in  10  c.c.  of  sterilized 
physiological  salt-solution.  The  more  thoroughly  it  is 
broken  up  the  more  accurate  will  be  the  results  of  the 
experiment.  Place  this  in  a  bath  of  boiling  water,  and  at 
the  end  of  one,  three,  five,  seven,  and  ten  minutes  make 
plates  upon  agar-agar  each  with  one  loopful  of  the  contents 
of  this  tube.  Are  the  results  on  the  plates  alike? 

Determine  the  exact  time  necessary  to  sterilize  objects, 
such  as  silk  or  cotton  threads,  on  which  anthrax  spores  have 
been  dried,  by  the  steam  method  and  by  the  hot-air  method. 


598     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

Prepare  a  bouillon  culture  from  the  blood  of  an  animal 
just  dead  of  anthrax.  After  this  has  been  in  the  incubator 
for  from  three  to  four  hours  subject  it  to  a  temperature  of 
55°  C.  for  ten  minutes.  At  the  end  of  this  time  make  plates 
from  it  and  also  inoculate  a  rabbit  subcutaneously  with  it. 
What  are  the  results?  Are  the  colonies  on  the  plates  in 
every  way  characteristic? 

Inoculate  six  Erlenmeyer  flasks  of  sterile  bouillon,  each 
containing  about  35  c.c.  of  the  medium,  from  the  blood  of  an 
animal  just  dead  of  anthrax. 

Place  these  flasks  in  the  incubator  at  a  temperature  of 
42.5°  C.  At  the  end  of  five,  ten,  fifteen,  twenty,  twenty-five, 
or  more  days  remove  a  flask.  Label  each  flask  as  it  is 
taken  from  the  incubator  with  the  exact  number  of  days 
that  it  has  been  at  the  temperature  of  42.5  C.  Study  each 
flask  carefully,  both  in  its  culture  peculiarities  and  in  its 
pathogenic  properties  when  employed  on  animals. 

Are  these  cultures  identical  in  all  respects  with  those  that 
have  been  kept  at  37°  C.? 

If  they  differ,  in  what  respect  is  the  difference  most  con- 
spicuous? 

Should  any  of  the  animals  survive  the  inoculations  made 
from  the  different  cultures  in  the  foregoing  experiment, 
note  carefully  which  one  it  is,  and  after  ten  to  twelve  days 
repeat  the  inoculation,  using  the  same  culture;  if  it  again 
survives,  inoculate  it  with  the  culture  preceding  the  one 
just  used  in  the  order  of  removal  from  the  incubator;  if 
it  still  survives,  inoculate  it  with  virulent  anthrax.  What  is 
the  result?  How  is  the  result  to  be  explained?  Do  the 
cultures  which  were  made  from  these  flasks  at  the  time  of 
their  removal  from  the  incubator  act  in  the  same  way  toward 


BACTERIUM  ANTHRACIS  599 

animals  as  the  organisms  growing  in  the  flasks?  Is  the 
action  of  each  of  these  cultures  the  same  for  mice,  guinea- 
pigs,  and  rabbits? 

Prepare  a  2  per  cent,  solution  of  sulphuric  acid  in  dis- 
tilled water;  suspend  in  this  a  number  of  anthrax  spores; 
at  the  end  of  three,  six,  and  nine  days  at  35°  C.  inoculate 
both  a  guinea-pig  and  a  rabbit.  Prepare  cultures  from  this 
suspension  on  the  third,  sixth,  and  ninth  days;  when  the 
cultures  have  developed  inoculate  a  rabbit  and  a  guinea- 
pig  from  the  culture  made  on  the  ninth  day.  Should  the 
animals  survive,  inoculate  them  again  after  three  or  four 
days  with  a  culture  made  on  the  sixth  day.  Do  the  results 
appear  in  any  way  peculiar? 


CHAPTER  XXVIII. 


The  Nitrifying  Bacteria — The  Bacillus  of  Tetanus — The  Bacillus  of  Malig- 
nant Edema — The  Bacillus  of  Symptomatic  Anthrax — Bacterium 
Welchii — Bacillus  Sporogenes. 


THE   NITRIFYING   BACTERIA. 

BY  the  employment  of  bacteriological  methods  in  the 
study  of  the  soil  much  light  has  been  shed  upon  the  cause 
and  nature  of  the  interesting  and  momentous  biological 
phenomena  there  constantly  in  progress.  Of  these,  the  one 
of  the  greatest  importance  comprises  those  changes  that 
accompany  the  widespread  process  of  disintegration  and 
decomposition,  to  which  reference  has  already  been  made. 
(See  Chapter  I.)  This  resolution  of  dead  complex  organic 
compounds  into  simpler  structures  assimilable  as  food  by 
growing  vegetation  is  dependent  upon  the  activities  of 
bacteria  located  in  the  superficial  layers  of  the  ground.  It 
is  not  a  simple  process,  brought  about  by  a  single,  specific 
species  of  bacteria,  but  represents  a  sequence  of  events  each 
of  which  probably-  results  from  the  activities  of  different 
species  or  groups  of  species,  working  alone  or  together.  Our 
knowledge  upon  the  subject  does  not  permit  of  the  following  in 
detail  of  the  manifold  alterations  undergone  by  dead  organic 
material,  but  we  do  know  that  much  of  it  is  ultimately 
converted  into  inorganic  matters  and  that  carbon  dioxide, 
ammonia  and  water  are  always  conspicuous  end  products. 
When  the  process  of  decomposition  occurs  in  the  soil  it 
does  not  cease  at  this  point,  but  we  find  still  further  altera- 
(600) 


THE  NITRIFYING  BACTERIA  601 

tions — alterations  having  to  do  more  particularly  with  the 
ammonia.  This  change  in  ammonia  is  characterized  by  the 
products  of  its  oxidation,  viz.,  by  the  formation  of  nitrous 
and  nitric  acids  and  their  salts;  this  is  not  a  result  of  the 
direct  action  of  atmospheric  oxygen  upon  the  ammonia, 
but  occurs  through  the  instrumentality  of  a  special  group 
of  saprophytes  known  generically  as  the  nitrifying  organ- 
isms. They  are  found  in  the  most  superficial  layers  of  the 
ground,  and  though  more  common  in  some  places  than  in 
others,  they  are,  nevertheless,  present  over  the  entire  surface 
of  the  earth.  The  most  conspicuous  example  of  the  func- 
tional activity  of  this  group  of  soil  organisms  is  seen  in  the 
immense  saltpeter-beds  of  Chili  and  Peru,  where,  by  the 
activities  of  these  microscopic  plants,  nitrates  are  produced 
from  the  ammonia  arising  from  the  decomposing  fecal 
evacuations  of  sea-fowls  and  from  decomposing  seaweeds 
in  such  enormous  quantities  as  to  form  a  source  of  supply 
of  crude  saltpeter  for  the  commercial  world.  A  more 
familiar  example  is  seen  in  the  decomposition  and  subse- 
quent nitrification  of  the  organic  matters  of  sewage  and 
other  fluid  wastes  of  organic  nature  in  the  process  of  puri- 
fication by  percolation  through  the  soil,  a  process  in  which 
it  is  possible  to  follow,  by  chemical  means,  the  organic 
matters  from  their  condition  as  such  to  their  ultimate  con- 
version into  inorganic  forms  of  ammonia,  nitrous  and  nitric 
acids.  In  fact,  the  same  breaking  down  and  building  up, 
resulting  ultimately  in  nitrification,  occurs  in  all  nitrogenous 
matters  that  are  deposited  upon  the  soil  and  allowed  to 
decay.  It  is  largely  through  this  means  that  growing  vege- 
tation obtains  the  nitrogen  necessary  for  the  nutrition  of  its 
tissues,  and  when  Viewed  from  this  standpoint  we  appre- 


602      APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

ciate  the  importance  of  this  process  to  all  life,  animal  as 
well  as  vegetable,  upon  the  earth. 

Under  special  circumstances  there  occurs  in  the  soil  a 
process  the  reverse  of  nitrification,  that  is,  a  reduction  of 
nitrates  and  nitrites  to  lower  compounds  and  ultimately 
to  free  gaseous  nitrogen.  This  so-called  "denitrification," 
while  the  result  of  bacterial  activity  is  not  dependent  upon 
such  specific  varieties  of  bacteria  as  is  nitrification.  For 
instance,  true  denitrification  is  known  to  be  an  attribute 
of  bacillus  coli  communis,  of  bacillus  fluorescens  lique- 
faciens,  of  bacillus  pyocyaneus,  and  of  bacillus  typhosus. 
While  this  group  of  species  ordinarily  develop  under 
free  access  of  oxygen  they  can  develop  without  it  and 
secure  their  necessary  oxygen  from  such  oxides  of  nitro- 
gen as  nitrates  and  nitrites,  thus  reducing  them.  It  seems 
probable  that  certain  products  o'f  bacterial  growth  have 
also  a  reducing  action  on  soil  nitrates.  Denitrification 
occurs  most  often  and  most  actively  in  soils  containing  an 
excess  of  undecomposed  organic  matter. 

In  addition  to  nitrification  and  denitrification  there  is 
seen  in  the  soil  a  phenomenon  resulting  in  "nitrogen  fixa- 
tion." In  some  instances  this  results  from  the  symbiotic 
activities  of  bacteria  and  higher  plants,. in  others  it  appears 
to  be  peculiar  to  certain  definite  species  of  bacteria  acting 
alone.  While  a  discussion  of  the  extreme  agricultural 
importance  of  these  phenomena  would  be  of  great  interest, 
yet  this  is  scarcely  the  place  to  undertake  it.1 

The  unusual  nature  of  the  nitrifying  bacteria,  demanding 
as  they  do  special  methods  for  their  cultivation,  renders 

1  See  Bacteria  in  Relation  to  Country  Life,  by  Lipman-MacMillan,  1911. 


THE  NITRIFYING  BACTERIA  603 

them  of  sufficient  technical  interest  to  justify — for  purposes 
of  illustration — a  more  or  less  detailed  description  of  one  of 
them. 

These  very  important  and  interesting  nitrifying  organisms, 
of  which  there  appear  to  be  several,  evade  all  efforts  to 
isolate  them  from  the  soils  and  to  cultivate  them  by  the 
methods  commonly  employed  in  bacteriological  work. 
They  can  be  successfully  studied  only  through  the  employ- 
ment of  special  media. 

The  organism  generally  known  as  the  nitro-monas  of 
Winogradsky  will  serve  as  an  illustration:  It  is  a  short, 
oval,  and  frequently  almost  spherical  cell.  It  reproduces 
by  segmentation  as  usual  for  bacteria,  but  there  is  little 
tendency  for  the  daughter-cells  to  adhere  together  or  to 
form  chains.  In  cultures  they  are  commonly  massed 
together,  by  a  gelatinous  material,  in  the  form  of  zooglea. 
It  does  not  form  spores,  and  is  probably  not  motile,  though 
Winogradsky  believes  he  has  occasionally  detected  it  in 
active  motion.  As  has  been  stated,  it  does  not  grow  upon 
ordinary  nutrient  media,  and  cannot,  therefore,  be  isolated 
by  the  means  commonly  employed  to  separate  different 
species  of  bacteria.  The  most  astonishing  property  of 
this  organism  is  its  ability  to  grow  and  perform  its  specific 
fermentative  function  in  solutions  devoid  of  organic  matter. 
It  is  believed  to  be  able  to  obtain  its  necessary  carbon  from 
carbon  dioxide.  For  its  isolation  and  cultivation  Wino- 
gradsky recommends  the  following  solution: 

Ammonium  sulphate 1  gram 

Potassium  phosphate 1  gram 

Pure  water    .  1000  c.c. 


604     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

To  each  flask  containing  100  c.c.  of  this  fluid  is  added  from 
0.5  to  1  gram  of  basic  magnesium  carbonate  suspended  in 
a  little  distilled  water  and  sterilized  by  boiling.  One  of  the 
flasks  is  then  to  be  inoculated  with  a  minute  portion  of  the 
soil  under  investigation,  and  after  four  or  five  days  a  small 
portion  is  to  be  withdrawn,  by  means  of  a  capillary  pipette, 
from  over  the  surface  of  the  layer  of  magnesium  carbonate 
and  transferred  to  a  second  flask,  and  similarly  after  four 
or  five  days  from  this  to  a  third  flask,  and  so  on.  As  this 
medium  does  not  offer  conditions  favorable  to  the  growth 
of  bacteria  requiring  organic  matter  for  their  development, 
those  that  were  originally  introduced  with  the  soil  quickly 
disappear,  and  ultimately  only  the  nitrifying  organisms 
remain.  These  are  seen  as  an  almost  transparent  film 
attached  to  the  clumps  and  granules  of  magnesium  carbonate 
on  the  bottom  of  the  flask. 

For  their  cultivation  upon  a  solid  medium  Winogradsky 
employs  a  mineral  gelatin,  the  gelatinizing  principle  of 
which  is  silicic  acid.  A  solution  of  from  3  to  4  per  cent, 
of  silicic  acid  in  distilled  water,  and  having  a  specific  gravity 
of  1.02,  remains  fluid  and  can  be  preserved  in  flasks  in  this 
condition.  (Kiihne.)  Gelatinization  occurs  after  the 
addition  of  certain  salts  to  such  a  solution,  and  will  be  more 
or  less  complete  according  to  the  proportion  of  salts  added. 
The  salts  that  have  given  the  best  results  and  the  method 
of  mixing  them  are  as  follows : 

Ammonium  sulphate 0.40  gram 

Magnesium  sulphate 0.05  gram 

Calcium  chloride trace. 

Potassium  phosphate 0.10  gram 

b\   Sodium  carbonate 0.6  to  0.90  gram 

Distilled  water  100.00  c.c. 


THE  NITRIFYING  BACTERIA  605 

The  sulphates  and  chloride  (a)  are  mixed  in  50  c.c.  of  the 
distilled  water,  and  the  phosphate  and  carbonate  (6)  in 
the  remaining  50  c.c.,  in  separate  flasks. 

Each  flask  is  then  sterilized  with  its  contents,  which  after 
cooling  are  mixed;  the  mixture  representing  the  solution 
of  mineral  salts  is  to  be  added  to  the  silicic  acid,  little  by 
little,  until  the  proper  degree  of  consistency  is  obtained  (that 
of  ordinary  nutrient  gelatin).  This  part  of  the  process  is 
best  conducted  in  a  culture-dish.  If  it  is  desired  to  separate 
the  colonies,  as  in  an  ordinary  plate,  the  inoculation  and 
mixing  of  the  material  introduced  must  be  done  before 
gelatinization  is  complete;  if  the  material  is  to  be  distributed 
over  only  the  surface  of  the  medium,  then  the  mixture 
must  first  be  allowed  to  solidify. 

By  the  use  of  the  silicate-gelatin  Winogradsky  has  isolated 
from  the  gelatinous  film  in  the  bottom  of  fluids  undergoing 
nitrification  a  bacillus  which  he  believes  to  be  associated 
with  the  nitro-monas  in  the  nitrifying  process. 

The  developments  in  this  field  of  study  are  of  such  breadth 
and  importance  that  they  can  scarcely  be  comprehended 
in  a  book  of  this  character.  For  particulars  the  reader  is 
referred  to  the  special  books  and  journals  dealing  with  the 
subject. 

In  addition  to  the  bacteria  concerned  in  the  various  trans- 
formation of  nitrogen,  there  are  occasionally  present  in  the 
soil  microorganisms  possessing  disease-producing  properties. 
Conspicuous  among  these  may  be  mentioned  the  bacillus 
of  malignant  edema  (vibrion  septique  of  the  French),  the 
bacillus  of  tetanus,  and  the  bacillus  of  symptomatic  anthrax 
(Rauschbrand  (Ger.);  charbon  symptomatique  (Fr.)).  It  is 
sometimes  due  to  the  presence  of  one  or  the  other  of  these 


606     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

organisms  that  wounds  to  which  soil  has  had  access  (crushed 
wounds  from  the  wheels  of  cars  or  wagons,  wounds  received 
in  agricultural  work,  gunshot  wounds,  etc.)  are  followed  by 
such  grave  consequences. 

BACILLUS   TETANI,    NICOLAEER,    1884. 

In  1884  Nicolaier  produced  tetanus  in  mice  and  rabbits 
by  the  subcutaneous  inoculation  of  particles  of  garden- 
earth,  and  demonstrated  that  the  pus  produced  at  the  point 
of  inoculation  was  capable  of  reproducing  the  disease  in 
other  mice  and  rabbits.  He  did  not  succeed  in  isolating 
the  organism  in  pure  culture.  In  1884  Carle  and  Rattone, 
and  in  1886  Rosenbach,  demonstrated  the  infectious  nature 
of  tetanus  as  it  occurs  in  man  by  producing  the  disease  in 
animals  by  inoculating  them  with  secretions  from  the 
wounds  of  individuals  affected  with  the  disease.  In  1889 
Kitasato  obtained  the  bacillus  of  tetanus  in  pure  culture, 
described  his  method  of  obtaining  it  and  detailed  its  bio- 
logical peculiarities  as  follows : 

Method  of  Obtaining. — Inoculate  several  mice  subcu- 
taneously  with  secretions  from  the  wound  of  a  case  of 
typical  tetanus.  This  material  usually  contains  not  only 
tetanus  bacilli,  but  other  organisms  as  well,  so  that  at 
autopsy,  if  tetanus  results,  there  may  be  more  or  less  sup- 
puration at  the  seat  of  inoculation  in  the  mice.  In  order 
to  separate  the  tetanus  bacillus  from  the  others  that  are 
present  the  pus  is  smeared  upon  the  surface  of  several 
slanted  blood  serum  or  agar-agar  tubes  and  placed  at  37° 
to  38°  C.  After  twenty-four  hours  all  the  organisms  will 
have  developed,  and  microscopic  examination  will  usually 


BACILLUS  TETANI  607 

reveal  the  presence  of  a  few  tetanus  bacilli,  recognizable 
by  their  shape,  viz.,  that  of  a  small  pin,  with  a  spore  repre- 
senting the  head.  After  forty-eight  hours  at  38°  C.  the 
culture  is  subjected  to  a  temperature  of  80°  C.  in  a  water- 
bath  for  from  three-quarters  to  one  hour.  At  the  end  of 
this  time  series  of  plates  or  Esmarch  tubes  of  slightly  alkaline 
gelatin  are  made  with  very  small  amounts  of  the  culture 
and  kept  in  an  atmosphere  of  hydrogen  (see  page  242). 
They  are  then  kept  at  from  18°  to  20°  C.,  and  at  the  end  of 
about  a  week  the  tetanus  bacillus  begins  to  appear  in  the 
form  of  colonies.  After  about  ten  days  the  colonies  should 
not  only  be  examined  microscopically,  but  each  colony 
that  has  developed  in  the  hydrogen  atmosphere  should  be 
obtained  in  pure  culture  and  again  grown  under  the  same 
conditions.  The  colonies  that  grow  only  without  oxygen, 
and  which  are  composed  of  the  pin-shaped  organisms, 
must  be  tested  upon  mice.  If  they  represent  growth  of  the 
tetanus  bacillus,  the  typical  clinical  manifestations  of  the 
disease  will  be  produced  in  these  animals. 

In  obtaining  the  organism  from  the  soil  much  difficulty 
is  experienced.  Here  are  encountered  a  number  of  spore- 
bearing  organisms  that  are  facultative  in  their  relation  to 
oxygen,  and  are  therefore  very  difficult  to  eliminate;  and 
there  is,  moreover,  one  in  particular  that,  like  the  tetanus 
bacillus,  forms  a  polar  spore.  This  spore  is,  however, 
much  more  oval  than  that  of  the  tetanus  bacillus,  and  gives 
to  the  organism  containing  it  more  the  shape  of  a  javelin 
(or  clostridium,  properly  speaking)  than  that  of  a  round- 
headed  pin,  the  characteristic  shape  of  the  spore-bearing 
tetanus  organism.  It  is  non-pathogenic,  and  grows  both 
with  and  without  oxygen,  and  should,  consequently,  not 


608     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

\ 

be  mistaken  for  the  latter  bacillus.  It  must  also  be  borne 
in  mind  that  there  are  occasionally  present  in  the  soil  still 
other  bacilli  which  form  polar  spores,  and  which,  when  in 
this  stage,  are  almost  identical  in  appearance  with  the  tetanus 
bacillus;  but  they  will  usually  be  found  to  differ  from  it 
in  their  relation  to  oxygen,  and  they  are  also  without  disease- 
producing  properties. 

FIG.  100 


Bacillus  teiani.     A,  vegetative  stage;    B,  spore-stage,  showing  pin-shapes. 

Morphology. — In  the  vegetating  stage  it  is  a  slender  rod 
with  rounded  ends.  It  may  appear  as  single  rods,  or,  in 
cultures,  as  long  threads.  It  is  motile,  though  not  actively 
so.  The  motility  is  rendered  somewhat  more  conspicuous 
by  examining  the  organism  upon  a  warm  stage. 

At  the  temperature  of  the  body  it  rapidly  forms  spores. 
These  are  round,  thicker  than  the  cell,  and  usually  occupy 
one  of  its  poles,  giving  to  the  rod  the  appearance  of  a  small 
pin.  (Fig.  100.)  When  in  the  spore-stage  it  is  not  motile. 


BACILLUS  TETANI 


609 


FIG.  101 


It  is  stained  by  the  ordinary  aniline  staining  reagents. 
It  retains  the  color  when  stained  by  Gram's  method. 

Cultural  Peculiarities. — It  is  an 
obligate  anaerobe,  and  cannot  be 
brought  to  development  under  access 
of  oxygen.  It  thrives  in  an  atmos- 
phere of  pure  hydrogen,  but  not  in 
one  of  carbonic  acid. 

It  grows  in  ordinary  nutrient 
gelatin  and  agar-agar  of  a  slightly 
alkaline  reaction.  Gelatin  is  slowly 
liquefied,  with  the  coincident,  pro- 
duction of  a  small  amount  of  gas. 
Blood  serum  is  not  liquefied  by  its 
growth. 

The  addition  to  the  media  of  from 
1.5  to  2  per  cent,  of  glucose,  0.1 
per  cent,  of  indigo-sodium  sulphate, 
or  5  per  cent,  by  volume  of  blue 
litmus  tincture  favors  its  growth. 

It  grows  well  in  alkaline  bouillon 
under  an  atmosphere  of  hydrogen. 

Under  artificial  conditions  it  may 
be  cultivated  through  numerous  gene- 
rations without  loss  of  virulence. 

Appearance  of  the  Colonies. — Colo- 
nies of  bacillus  tetani  on  gelatin  under 
an  atmosphere  of  hydrogen  have, 


Colonies  of  the  tetanus 
bacillus  four  days  old, 
made  by  distributing  the 

in  their  early  stages,  somewhat  the    organisms  through  a  tube 
of   the   colonies   of   the 


appearance 

common    bacillus    subtilis    in    their 

earliest    stages,    viz.,   they    have    a 

39 


nearly  filled  with  glucose- 
gelatin.  Cultivation  in 
an  atmosphere  of  hydro- 
gen. (From  Frankel  and 
Pfeiffer.) 


610     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

dense,  felt-like  center  surrounded  by  a  fringe  of  delicate 
radii.  The  liquefaction  is  so  slow  that  the  appearance  is 
retained  for  a  relatively  long  time,  but  eventually  becomes 
altered.  In  very  old  colonies  the  entire  mass  is  made 
up  of  a  number  of  distinct  threads  that  give  it  the  ap- 
pearance of  a  common  mould.  (See  Fig.  101.) 

In  stab-cultures  made  in  tubes  about  three-quarters  filled 
with  gelatin  growth  begins  at  about  1.5  to  3  cm.  below  the 
surface,  and  gradually  assumes  the  appearance  of  a  cloudy, 
linear  mass,  with  prolongations  radiating  into  the  gelatin 
from  all  sides.  Liquefaction  with  coincident  gas-production 
results,  and  may  reach  almost  to  the  surface  of  the  gelatin. 

Relation  to  Temperature  and  to  Chemical  Agents. — It  grows 
best  at  a  temperature  of  from  36°  to  38°  C.;  gelatin  cultures 
kept  at  from  20°  to  25°  C.  begin  to  grow  after  three  or  four 
days.  In  an  atmosphere  of  hydrogen  at  from  18°  to  20°  C. 
growth  does  not  usually  occur  before  one  week.  No  growth 
occurs  below  14°  C.  At  the  temperature  of  the  body  spores 
are  formed  in  cultures  in  about  thirty  hours,  whereas  in 
gelatin  cultures  at  from  20°  to  25°  C.  they  do  not  usually 
appear  before  a  week,  when  the  lower  part  of  the  gelatin 
is  quite  fluid. 

Spores  of  the  tetanus  bacillus  when  dried  upon  bits  of 
thread  over  sulphuric  acid  in  the  desiccator  and  subse- 
quently kept  exposed  to  the  air,  retain  their  vitality  and 
virulence  for  a  number  of  months.  Their  vitality  is  not 
destroyed  by  an  exposure  of  one  hour  to  80°  C.;  on  the 
other  hand,  an  exposure  of  five  minutes  to  100°  C.  in  the 
steam  sterilizer  kills  them.  They  resist  the  action  of  5  per 
cent,  carbolic  acid  for  ten  hours,  but  succumb  when  exposed 
to  it  for  fifteen  hours.  In  the  same  solution,  plus  0.5  per 
cent,  of  hydrochloric  acid,  they  are  no  longer  active  after 


BACILLUS  TETANI  611 

two  hours.  They  are  killed  when  acted  upon  for  three 
hours  by  corrosive  sublimate,  1  :  1000,  and  in  thirty  minutes 
by  the  same  solution  plus  0.5  per  cent,  of  hydrochloric  acid. 

Action  upon  Animals. — After  subcutaneous  inoculation 
of  mice  with  minute  portions  of  a  pure  culture  of  this 
organism  tetanus  develops  in  twenty-four  hours  and  ends 
fatally  in  from  two  to  three  days.  Rats,  guinea-pigs,  and 
rabbits  are  similarly  affected,  but  only  by  larger  doses  than 
are  required  for  mice,  the  fatal  dose  for  a  rabbit  being  from 
0.3  to  0.5  c.c.  of  a  well-developed  bouillon  culture.  The 
period  of  incubation  for  rats  and  guinea-pigs  is  twenty-four 
to  thirty  hours,  and  for  rabbits  from  two  to  three  days. 
Pigeons  are  but  slightly,  if  at  all,  susceptible. 

The  tetanic  convulsions  always  appear  first  in  the  parts 
nearest  the  seat  of  inoculation,  and  subsequently  become 
general. 

At  autopsies  upon  animals  that  have  succumbed  to 
inoculations  with  pure  cultures1  of  bacillus  tetani  there  is 
little  to  be  seen  by  either  macroscopic  or  microscopic  exami- 
nation, and  cultures  from  the  site  of  inoculation  are  often 
negative  in  so  far  as  finding  the  tetanus  bacillus  is  concerned. 
At  the  site  of  inoculation  there  is  usually  only  a  hyperemic 
condition.  In  uncomplicated  cases  there  is  no  suppuration. 
The  internal  organs  do  not  present  any  macroscopic  change, 
and  culture-methods  of  examination  show  them  to  be  free 
from  bacteria.  The  death  of  the  animal  results  from  the 
absorption  of  a  soluble  poison,  either  produced  by  the  bac- 
teria at  the  site  of  inoculation  or,  which  seems  more  probable, 
produced  by  the  bacteria  in  the  culture  from  which  they  are 

1  Animals  and  human  beings  that  have  become  infected  with  this  organism 
in  the  ordinary  way  commonly  present  a  condition  of  suppuration  at  the 
site  of  infection;  this  is  not  due,  however,  to  the  tetanus  bacillus,  but 
to  other  bacteria  that  gained  access  to  the  wound  at  the  time  of  infection. 


612     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

obtained  and  introduced  with  them  into  the  tissues  of  the 
animal  at  the  time  of  inoculation.  In  support  of  the  latter 
hypothesis;  mice  have  been  inoculated  with  pure  cultures 
of  this  organism;  after  one  hour  the  point  at  which  the 
inoculation  was  made  was  excised  and  the  tissues  cauterized 
with  a  hot  iron;  notwithstanding  the  short  time  during 
which  the  organisms  were  in  contact  with  the  tissues  and 
the  subsequent  radical  treatment,  the  animals  died  after 
the  usual  interval  and  with  the  typical  symptoms  of  tetanus. 

The  poison  produced  by  the  tetanus  bacillus,  and  to 
which  the  symptoms  of  the  disease  are  due,  has  been  isolated 
and  subjected  to  detailed  study;  some  of  its  toxic  peculiari- 
ties, as  given  by  Kitasato,  are  as  follows:1 

"When  cultures  of  this  organism  are  robbed  of  their 
bacteria  by  filtration  through  porcelain  the  filtrate  contains 
the  soluble  poison,  and  is  capable,  when  injected  into  animals, 
of  causing  tetanus. 

"Inoculations  of  other  animals  with  bits  of  the  organs 
of  the  animal  dead  from  the  action  of  the  tetanus  toxin 
produce  no  result;  but  similar  inoculations  with  the  blood 
or  with  the  serous  exudate  from  the  pleural  cavity  always 
result  in  the  appearance  of  tetanus.  The  poison  is,  there- 
fore, largely  present  in  the  circulating  fluids. 

"  The  greatest  amount  of  poison  is  produced  by  cultivation 
in  fresh  neutral  bouillon  of  a  very  slightly  alkaline  reaction. 

"The  activity  of  the  poison  is  destroyed  by  an  exposure 
of  one  and  one-half  hours  to  55°  C.;  of  twenty  minutes  to 
60°  C.;  and  of  five  minutes  to  65°  C. 

"By  drying  at  the  temperature  of  the  body  under  access 
of  air  the  poison  .is  destroyed;  but  by  drying  at  the  ordinary 

iZeitschrift  fur  Hygiene,  1891,  Bd.  x,  S.  267. 


BACILLUS  TETANI  613 

temperature  of  the  room,  or  at  this  temperature  in  the  desic- 
cator over  sulphuric  acid,  it  is  not  destroyed. 

"Diffuse  daylight  diminishes  the  intensity  of  the  poison. 
Its  intensity  is  preserved  when  kept  in  the  dark. 

"Direct  sunlight  robs  it  of  its  poisonous  properties  in 
from  fifteen  to  eighteen  hours. 

"  Its  activity  is  not  diminished  by  diluting  a  fixed  amount 
with  water  or  nutrient  bouillon. 

"Mineral  acids  and  strong  alkalies  lessen  its  intensity." 

The  chemical  nature  of  this  poison  is  not  positively  known, 
but  its  designation  "Toxalbumen"  is  probably  a  misnomer, 
for  its  reactions  do  not  warrant  its  classification  with  the 
albumins  in  the  sense  in  which  the  word  is  commonly  used. 
When  obtained  in  a  concentrated  form,  its  toxic  properties 
are  seen  to  be  altered  by  acids,  by  alkalies,  by  sulphuretted 
hydrogen,  and  by  temperatures  above  70°  C.  Even  when 
carefully  protected  from  light,  moisture  and  air,  it  gradually 
becomes  diminished  in  strength,  doubtless  due  to  the  forma- 
tion of  "toxons"  and  "toxoids,"  analogous  to  those  observed 
by  Ehrlich  in  deteriorating  diphtheria  toxin.  When  freshly 
prepared  its  potency  is  almost  incredible,  0.00005  milligrams 
being  sufficient  to  cause  fatal  tetanus  in  a  mouse  weighing 
15  grams. 

The  studies  of  Madsen1  demonstrate  it  to  consist  of  two 
physiologically  distinct  intoxicating  compounds;  the  one, 
a  solvent  of  erythrocytes — a  "tetanolysin;"  the  other,  a 
specific  irritant  which,  through  its  influence  upon  the  central 
nervous  system,2  accounts  for  the  phenomena  by  which 

1  Ueber  Teanolysin,  Zeitschrift  fur  Hygiene  und  Infektionskrankheiten, 
1899,   Bd.   xxxii,   S.   214. 

2  See  paper  by  Wassermann  and  Takaki,   Berliner  klinische  Wochen- 
schrift,  1898,  No.  1,  S.  5. 


614      APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

tetanus  is  characterized;  to  this  latter  the  designation 
"  tetanospasmin"  is  given.  Madsen's  observations,  further- 
more, confirm  the  deductions  of  Ehrlich  concerning  the 
molecular  structure  of  bacterial  toxins  in  general,  to  the 
effect  that  the  molecule  of  tetanolysin,  like  that  of  diph- 
theria toxin,  is  a  complex  of  at  least  two  physiologically 
unlike  groups;  the  one,  characterized  by  its  marked  com- 
bining tendencies  (for  antitoxin),  the  so-called  haptophore 
group;  the  other,  distinguished  for  its  intoxicating  quality, 
the  so-called  toxophore  group. 

Tetanus  Antitoxin. — The  principles  involved  in  the  induction 
of  the  antitoxic  state  against  diphtheria  are  likewise  applicable 
to  tetanus;  in  fact,  the  fundamental  observations  upon  the 
generation  of  antitoxin  in  the  living  animal  body  were  made  in 
the  course  of  studies  on  tetanus;  they  were  subsequently  ap- 
plied to  the  study  of  diphtheria,  with  the  results  already  noted. 
It  is  needless  to  enter  here  upon  the  details  essential  to  the 
production  of  tetanus  antitoxin;  to  all  intents  and  purposes, 
they  are  identical  with  those  given  in  the  section  on  diph- 
theria. Briefly  stated,  animals  may  be  rendered  immune 
from  tetanus  by  the  repeated  injection  of  gradually  increas- 
ing non- fatal  doses  of  tetanus  toxin;  when  immunity  is 
established,  the  circulating  blood  contains  a  body,  anti- 
toxin, that  combines  directly  with  tetanus  toxin  in  a  test- 
tube,  and  thereby  renders  it  physiologically  inactive  (non- 
intoxicating);  and  the  serum  of  the  immune  animal  is  not 
only  capable  of  protecting  non-immune,  susceptible  animals 
from  the  poisonous  action  of  tetanus  toxin  (within  limits), 
but  also  against  the  effects  of  the  living  tetanus  bacillus  as 
well. 

Tetanus  antitoxin,  though  the  first  antitoxin  discovered 


BACILLUS  TETANI  615 

and  frequently  employed  in  the  treatment  of  tetanus,  has 
not  yielded  as  brilliant  results  as  those  obtained  with  diph- 
theria antitoxin.  There  are  good  reasons  why  tetanus 
antitoxin  may  never  be  expected  to  yield  such  satisfactory 
results  as  does  diphtheria  antitoxin.  Diphtheria  infection 
can  be  recognized  by  bacteriological  methods  and  the  anti- 
toxin administered  long  before  very  marked  constitutional 
symptoms  have  developed,  and  consequently  long  before 
the  diphtheria  toxin  has  had  time  to  bring  about  serious 
tissue  alterations.  In  tetanus  it  is  impossible  to  make  such 
a  definite  bacteriological  examination,  and  very  frequently 
the  first  suggestion  of  the  disease  is  the  twitching  of  the 
muscles,  the  antecedent  sign  of  the  tetanic  convulsions. 
When  these  clinical  manifestations  have  developed  in  tetanus 
there  is  already  very  serious  involvement  of  the  central 
nervous  system. 

In  the  use  of  tetanus  antitoxin  it  is  advisable  to  employ 
it  as  early  as  possible  and  to  give  repeated  doses  until  the 
symptoms  are  relieved.  Whether  the  subdural  adminis- 
tration of  the  antitoxin  will  be  of  greater  value  than  the 
subcutaneous  administration  is  as  yet  undecided. 

A  great  deal  of  benefit  results,  from  the  administration 
of  tetanus  antitoxin  as  a  prophylactic  in  the  treatment  of 
wounds  in  which  infection  by  the  tetanus  bacillus  is  possible. 
The  prophylactic  injection  of  the  tetanus  antitoxin  in  these 
cases,  however,  should  always  be  accompanied  by  approved 
surgical  treatment  of  the  wound,  and  under  these  conditions 
it  is  more  or  less  doubtful  which  of  these  measures  is  of 
the  greater  value,  but  experience  seems  to  indicate  that  the 
antitoxin  has  a  distinct  prophylactic  influence  in  these  cases. 


616     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 


BACILLUS    EDEMATIS,    LIBORIUS,    1886. 

The  bacillus  of  malignant  edema,  also  known  as  vibrion 
septique,  is  another  pathogenic  form  almost  everywhere 
present  in  the  soil.  In  certain  respects  it  is  a  little  like 
bacterium  anthracis,  and  was  at  one  time  confounded  with 
it;  but  it  differs  in  the  marked  peculiarity  of  being  a  strict 
anaerobe.  It  was  first  observed  by  Pasteur,  but  it  was  not 
until  later  that  Koch,  Laborious,  Kitt,  and  others  described 
its  peculiarities  in  detail.  It  can  often  be  obtained  by 
inserting  under  the  skin  of  rabbits  or  guinea-pigs  small 
portions  of  garden-earth,  street-dust,  or  decomposing 
organic  substances.  There  results  a  widespread  edema, 
with  more  or  less  gas-production  in  the  tissues.  In  the 
edematous  fluid  about  the  site  of  inoculation  the  organism 
under  consideration  may  be  detected.  (Fig.  102,  A.) 

It  is  a  rod  about  3  to  3.5/z  long  and  from  1  to  1.1  ju  thick 
— i.  e.,  it  is  about  as  long  as  bacterium  anthracis,  but  is  a 
trifle  more  slender.  It  is  usually  found  in  pairs,  joined  end 
to  end,  but  may  occur  as  longer  threads;  particularly  is 
this  the  case  in  cultures.  When  in  pairs  the  ends  that 
approximate  are  squarely  cut,  while  the  distal  extremities 
are  rounded.  When  occurring  singly  both  ends  are  rounded. 
(How  does  it  differ  in  this  respect  from  bacterium  anthracis?) 
It  is  slowly  motile,  and  its  flagella  are  located  both  at  the 
ends  and  along  the  sides  of  the  rod.  It  forms  spores  that 
are  usually  located  in  or  near  the  middle  of  the  cells,  causing 
frequently  a  swelling  at  the  points  at  which  they  are  located 
and  giving  to  the  cell  a  more  or  less  oval,  spindle,  or  lozenge 
shape.  (Fig.  102,  B.) 

It  is  an  obligate  anaerobe,  growing  on  all  the  ordinary 


BACILLUS  EDEMATIS 


617 


media,  but  not  with  access  of  oxygen.    It  grows  well  in  an 

atmosphere  of  hydrogen.     It  causes  liquefaction  of  gelatin. 

In  tubes  containing  about  20  to  30  c.c.  of  gelatin  that 

has  been  liquefied,  inoculated  with  a  small  amount  of  the 


FIG.  102 


a 


'*• 


'•* 


Bacillus  edematis.     A,  edema-fluid,  from  site  of  inoculation  of  guinea-pig, 
showing  long  and  short  threads;    B,  spore-formation,  from  culture. 

culture,  and  then  rapidly  solidified  in  ice-water,  growth 
appears  in  the  form  of  isolated  colonies  at  or  near  the  bottom 
of  the  tube  in  from  two  to  three  days  at  20°  C.  These 
colonies,  when  of  from  0.5  to  1  mm.  in  diameter,  appear  as 
spheres  filled  with  clear  liquid,  and  are  difficult,  for  this 


618     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 


FIG.  103 


reason,  to  detect.  (Fig.  103.)  As  they  gradually  increase 
in  size  the  contents  of  the  spheres  become  cloudy  and 
marked  by  fine  radiating  stripes,  easily 
to  be  detected  with  the  aid  of  a  small 
hand-lens.  In  deep  stab-cultures  in  agar- 
agar  and  in  gelatin  development  occurs 
only  along  the  track  of  puncture,  at  a 
distance  below  the  surface.  Growth  is 
frequently  accompanied  by  the  produc- 
tion of  gas-bubbles. 

It  causes  rapid  liquefaction  of  blood 
serum,  with  production  of  gas-bubbles, 
and  in  two  or  three  days  the  entire 
medium  may  have  become  converted 
into  a  yellowish  semifluid  mass. 

The  most  satisfactory  results  in  the 
study  of  the  colonies  are  obtained  by  the 
use  of  plates  of  nutrient  agar-agar  kept 
in  a  chamber  in  which  all  oxygen  has 
been  replaced  by  hydrogen.  The  colo- 
nies appear  as  dull  whitish  points,  irreg- 
ular in  outline,  and  when  viewed  with  a 
low-power  lens  are  seen  to  be  marked  by 
a  net-work  of  branching  and  interlacing 
lines  that  radiate  in  an  irregular  way 
from  the  center  toward  the  periphery. 

It  grows  well  at  the  ordinary  tempera- 
ture of  the  room,  but  reaches  its  highest 
development  at  the  temperature  of  the 
body. 

It  stains  readily  with  the  ordinary  aniline  dyes.    It  does 
not  stain  by  Gram's  method. 


Colonies  of  the  ba- 
cillus of  malignant 
edema  in  deep  gela- 
tin culture. '  (After 
FrankelandPfeiffer.) 


BACILLUS  EDEMATIS  619 

Pathogenesis. — The  animals  known  to  be  susceptible  to 
inoculation  with  this  organism  are  man,  horses,  calves, 
dogs,  goats,  sheep,  pigs,  chickens,  pigeons,  rabbits,  guinea- 
pigs,  and  mice.  Cases  are  recorded  in  which  men  and  horses 
have  developed  the  disease  after  injuries,  doubtless  due  to 
the  introduction  into  the  wound,  at  the  time,  of  soil  or  dust 
containing  the  organism. 

If  one  introduce  into  a  pocket  beneath  the  skin  of  a  sus- 
ceptible animal  about  as  much  garden-earth  as  can  be  held 
upon  the  point  of  a  penknife,  the  animal  frequently  dies  in 
from  twenty-four  to  forty-eight  hours.  The  most  conspic- 
uous result  found  at  autopsy  is  a  wide-spread  edema  at 
and  about  the  site  of  inoculation.  The  edematous  fluid  is 
in  some  places  clear,  while  at  others  it  may  be  stained  with 
blood;  it  is  usually  rich  in  bacilli  (Fig.  102,  A)  and  contains 
gas-bubbles.  Of  the  internal  organs  only  the  spleen  shows 
much  damage.  It  is  large,  dark  in  color,  and  contains 
numerous  bacilli.  If  the  autopsy  be  made  immediately 
after  death,  bacilli  are  rarely  found  in  the  blood  of  the 
heart;  but  if  deferred  for  several  hours,  the  organisms  will 
be  found  in  this  locality  also,  a  fact  that  speaks  for  their 
multiplication  in  the  body  after  death.  At  the  moment  of 
death  they  are  present  in  varying  numbers  in  all  the  internal 
viscera  and  on  the  serous  surfaces  of  the  organs. 

Of  all  animals  mice  are  probably  the  most  susceptible 
to  the  action  of  this  organism,  and  it  is  not  rare  to  find  it 
in  the  heart's  blood,  even  immediately  after  death.  They 
die,  as  a  result  of  these  inoculations,  in  from  sixteen  to 
twenty  hours. 

When  a  pure  culture  is  used  for  inoculation  a  relatively 
large  amount  must  be  employed,  and  this  should  be  deposited 
in  the  subcutaneous  tissues  at  some  distance  from  the  surface. 


620      APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

In  continuing  the  inoculations  from  animal  to  animal 
small  portions  of  organs  or  a  few  drops  of  the  edema-fluid 
should  be  used.  The  inoculation  may  also  be  successfully 
made  by  introducing  into  a  pocket  in  the  skin  bits  of  steril- 
ized thread  or  paper  upon  which  cultures. have  been  dried. 

The  methods  for  obtaining  the  organism  in  pure  culture, 
from  the  cadaver  of  an  animal  that  has  succumbed  to  infec- 
tion by  the  bacillus  of  malignant  edema,  are  in  all  essential 
respects  the  same  as  those  given  for  obtaining  cultures 
from  tissues  in  general;  but  it  must  be  remembered  that 
the  organism  is  a  strict  anaerobe,  and  will  not  grow  under 
the  influence  of  oxygen.  (See  methods  of  cultivating 
anaerobic  species.) 

In  certain  superficial  respects  this  bacillus  suggests,  as 
said  above,  bacterium  anthracis,  but  differs  from  it  in  so 
many  important  details  that  there  is  no  excuse  for  con- 
founding the  two. 

NOTE. — From  what  has  been  said  of  this  organism,  what 
are  the  most  important  differential  points  between  it  and 
bacillus  anthracis?  Inoculate  several  mice  with  small  por- 
tions of  garden-earth  and  street-dust.  Isolate  the  organism 
that  agrees  most  nearly  with  the  description  here  given  for 
the  bacillus  of  malignant  edema.  Compare  its  morpholog- 
ical, biological,  and  pathogenic  peculiarities  with  those  of 
bacillus  anthracis  under  similar  circumstances;  especially 
its  appearance  in  the  tissues  and  fluids. 

Still  another  pathogenic  organism  that  may  be  present 
in  the  soil  is : — 


BACILLUS  CHAUVEI 


621 


BACILLUS   CHAUVEI,   ARLOING,   CORNEVIN,   AND 
THOMAS,    1887. 

SYNONYMS:  The  bacillus  of  symptomatic  anthrax — Bacterie  du  cha'rbon 
symptomatique  (Fr.) — Bacillus  des  rauschbrand  (Ger.). 

It  is  the  organism  concerned  in  the  production  of  the 
disease  of  young  cattle  and  sheep  commonly  known  as 
"black  leg/'  "quarter  evil,"  and  "quarter  ill,"  a  disease 

FIG.  104 


Bacillus  of  symptomatic  anthrax.     A,  vegetative   stage — gelatin   culture 
B,  spore-forms — agar-agar  culture. 


that  prevails  in  certain  localities  during  the  warm  months, 
and  which  is  characterized  by  a  peculiar  emphysematous 
swelling  of  the  muscular  and  subcutaneous  cellular  tissues 
over  the  quarters.  The  muscles  and  cellular  tissues  at  the 
points  affected  are  seen  on  section  to  be  saturated  with 
bloody  serum,  and  the  muscles  particularly  are  of  a  dark, 
almost  black  color.  In  these  areas,  in  the  bloody  transu- 


622     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 


FIG.  105 


dates  of  the  serous  cavities,  in  the  bile,  and,  after  death, 
in  the  internal  organs,  the  organism  to  be  described  can 
always  be  detected.  It  is  manifest  from 
this  that  the  soil  of  localities  over  which 
infected  herds  are  grazing  may  readily 
become  contaminated  through  a  variety 
of  channels,  and  thus  serve  as  a 
source  of  further  dissemination  of  the 
disease. 

The  organism  was  first  observed  by 
Feser,  and  subsequently  by  Bollinger 
and  others.  The  most  complete  de- 
scription of  its  morphological  and  bio- 
logical peculiarities  is  that  of  Kitasato.1 
The  following  is  from  Kitasato's  contri- 
butions: it  is  an  actively  motile  rod 
about  3  to  5/z  long  by  0.5  to  0.6/z 
thick.  It  has  rounded  ends,  and,  as  a 
rule,  is  seen  singly,  though  now  and  then 
pairs  joined  end  to  end  may  occur.  It 
has  no  tendency  to  form  very  long 
threads.  (Fig.  104,  A) 

It  forms  spores,  and  when  in  this  stage 
is  seen  to  be  slightly  swollen  at  or  near 
Colonies    of    the     one  of  its  poles,  the    location  in  which 

the    SPMe    USUally    aPPearS'       ^-     104, 

B.)      It  is  markedly  prone   to    undergo 

,  ...  ,  ,      .         ,    ,. 

degenerative    changes,     and    involution- 
forms   are   commonly    seen    not  only  in 

fresh   cultures,  but  in  «the  tissues   of   affected    animals  as 

well. 

iZeitschrift  fur  Hygiene.  Bd.  vi,  S.  105;  13d.  viii,  S.  55. 


deep  gelatin  culture. 

(After  Frankel    and 

Pfeiffer.) 


BACILLUS  CHAUVEI  623 

Though  actively  motile  when  in  the  vegetative  stage,  it, 
like  all  other  motile  spore-forming  bacilli,  loses  this  property 
and  becomes  motionless  when  spores  are  forming. 

It  is  strictly  anaerobic  and  cannot  be  cultivated  in  an 
atmosphere  in  which  free  oxygen  is  present.  It  grows  best 
under  hydrogen,  and  does  not  grow  under  carbonic  acid. 

The  media  most  favorable  to  its  growth  are  those  con- 
taining glucose  (1.5  to  2  per  cent.),  glycerin  (4  to  5  per 
cent.),  or  some  other  reducing-body,  such  as  indigo-sodium 
sulphate,  sodium  formate,  etc. 

When  cultivated  upon  gelatin  plates  in  an  atmosphere  of 
hydrogen  the  colonies  appear  as  irregular,  slightly  lobu- 
lated  masses.  After  a  short  time  liquefaction  of  the  gelatin 
occurs  and  the  colony  presents  a  dark,  dense,  lobulated  and 
broken  center,  surrounded  by  a  much  more  delicate,  fringe- 
like  zone. 

When  distributed  through  a  deep  layer  of  liquefied  gelatin 
that  is  subsequently  solidified  colonies  develop  at  only  the 
lower  portions  of  the  tube.  The  single  colonies  appear  as 
discrete  globules  that  cause  rapid  liquefaction  of  the  gelatin, 
and  ultimately  coalesce  into  irregular,  lobulated  liquid 
areas.  In  some  of  the  larger- colonies  an  ill-defined,  concen- 
tric arrangement  of  alternate  clear  and  cloudy  zones  can 
be  made  out  (Fig.  105). 

In  deep  stab-cultures  in  gelatin  growth  begins  after  about 
two  or  three  days  at  20°  to  25°  C.  It  begins  usually  at 
about  one  or  two  centimeters  below  the  surface,  and  causes 
slow  liquefaction  at  and  around  the  track  of  its  development. 
During  its  growth  gas-bubbles  are  produced. 

In  deep  stab-cultures  in  agar-agar  at  37°  to  38°  C.  growth 
begins  in  from  twenty-four  to  forty-eight  hours,  also  at 
about  one  or  two  centimeters  below  the  surface,  and  is 


624     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

accompanied  by  the  production  of  gas-bubbles.  There  is 
produced  at  the  same  time  a  peculiar,  penetrating  odor 
somewhat  suggestive  of  that  of  rancid  butter.  Under  these 
conditions  spores  are  formed  after  about  thirty  hours. 

It  grows  well  in  bouillon  of  very  slightly  acid  reaction 
under  hydrogen,  but  does  not  retain  its  virulence  for  so 
long  a  time  as  when  cultivated  upon  solid  media.  In  this 
medium  it  develops  in  the  form  of  white  flocculi  that  sink 
ultimately  to  the  bottom  of  the  glass  and  leave  the  super- 
natant fluid  quite  clear.  If  the  vessel  be  now  gently  shaken, 
these  delicate  flakes  are  distributed  homogeneously  through 
it.  In  bouillon  cultures  there  is  often  seen  a  delicate  ring 
of  gas-bubbles  round  the  point  of  contact  of  the  tube  and 
the  surface  of  the  bouillon.  There  is  produced  also  a  pecu- 
liar, penetrating,  sour  or  rancid  odor. 

It  grows  best  at  the  body-temperature — i.  e.,  from  37°  to 
38°  C. — but  can  also  be  brought  to  development  at  from 
16°  to  18°  C.  Below  14°  C.  no  growth  is  seen.  Spore- 
formation  appears  much  sooner  at  the  higher  than  at  the 
lower  temperatures.  When  its  spores  are  dried  upon  bits 
of  thread  in  the  desiccator  over  sulphuric  acid,  and  then 
kept  under  ordinary  conditions,  they  retain  their  vitality 
and  virulence  for  many  months.  Similarly,  bits  of  flesh 
from  the  affected  areas  of  animals  dead  of  this  disease,  when 
completely  dried,  are  seen  to  retain  for  a  long  time  the  power 
of  reproducing  the  disease.  The  spores  are  tolerably  resist- 
ant to  the  influence  of  heat:  when  subjected  to  a  tempera- 
ture of  80°  C.  for  one  hour  their  virulence  is  not  affected, 
but  an  exposure  to  100°  C.  for  five  minutes  destroys  them. 
They  are  also  seen  to  be  somewhat  resistant  to  the  action 
of  chemicals:  when  exposeo!  to  5  per  cent,  carbolic  acid 
they  retain  their  disease-producing  properties  for  about 


BACILLUS  CHAUVEI  625 

ten  hours,  tvhereas  the  vegetative  forms  are  destroyed  in 
from  three  to  five  minutes;  in  corrosive  sublimate  solution 
of  the  strength  of  1  : 1000  the  spores  are  killed  in  two  hours. 

When  gelatin  cultures  are  examined  microscopically  the 
organisms  are  usually  seen  as  single  rods  with  rounded  ends. 
When  cultivated  in  agar-agar  at  a  higher  temperature 
spores  are  formed  after  a  short  time;  the  spores  are  oval, 
slightly  flattened  on  their  sides,  thicker  than  the  bacilli, 
and,  as  stated,  frequently  occupy  a  position  inclining  to 
one  of  the  poles  of  the  bacillus,  though  they  are  as  often 
seen  in  the  middle. 

Bacilli  containing  spores  are  usually  clubbed  or  spindle 
shape. 

This  bacillus  stains  readily  with  the  ordinary  aniline 
dyes.  It  is  decolorized  by  Gram's  method.  Its  spores 
may  be  stained  by  the  methods  usually  employed  in  spore- 
staining. 

Pathogenesis. — When  susceptible  animals,  especially 
guinea-pigs,  are  inoculated  in  the  deeper  subcutaneous 
cellular  tissues  with  pure  cultures  of  this  organism,  or  with 
bits  of  tissue  from  the  affected  area  of  another  animal  dead 
of  the  disease,  death  ensues  in  from  one  to  two  days.  It 
is  preceded  by  rise  of  temperature,  loss  of  appetite,  and 
general  indisposition.  The  site  of  inoculation  is  swollen 
and  painful,  and  drops  of  bloody  serum  may  sometimes  be 
seen  exuding  from  it.  At  autopsy  the  subcutaneous  cellular 
tissues  and  underlying  muscles  present  a  condition  of 
emphysema  and  extreme  edema.  The  edematous  fluid  is 
often  blood-stained  and  the  muscles  are  of  a  blackish  or 
blackish-brown  color.  The  lymphatic  glands  are  markedly 
hyperemic.  The  internal  viscera  present  but  little  altera- 
tion visible  to  the  naked  eye.  In  the  blood-stained  serous 
40 


626      APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

fluid  about  the  point  of  inoculation  short  bacilli  are  present 
in  large  numbers.  These  often  present  slight  swellings  at 
the  middle  or  near  the  end.  They  are  not  seen  as  threads, 
but  lie  singly  in  the  tissues.  Occasionally  two  will  be  seen 
joined  end  to  end.  If  the  autopsy  be  made  immediately 
after  death,  these  organisms  may  not  be  detected  in  the 
internal  organs;  but  if  not  made  until  after  a  few  hours, 
they  will  be  found  there  also.  In  recent  autopsies  only 
vegetative  forms  of  the  organism  may  be  found;  but  later 
(in  from  twenty  to  twenty-four  hours)  spore-bearing  rods 
may  be  detected.  (How  does  this  compare  with  bacterium 
anthracisf)  By  successive  inoculations  of  susceptible 
animals  with  serous  fluid  from  the  site  of  inoculation  of  the 
dead  animal  the  disease  may  be  reproduced. 

Cattle,  sheep,  goats,  guinea-pigs,  and  mice  are  susceptible 
to  infection  with  this  organism,  and  present  the  conditions 
above  described;  whereas  horses,  asses,  and  white  rats 
present  only  local  swelling  at  the  site  of  inoculation.  Swine, 
dogs,  cats,  rabbits,  ducks,  chickens,  and  pigeons  are,  as  a 
rule,  naturally  immune  from  the  disease. 

Though  closely  simulating  the  bacillus  of  malignant 
edema  in  many  of  its  peculiarities,  this  organism  can 
nevertheless,  be  readily  distinguished  from  it.  It  is  smaller; 
it  does  not  develop  into  long  threads  in  the  tissues;  it  is 
more  actively  motile,  and  forms  spores  more  readily  in  the 
tissues  of  the  animal  than  does  the  bacillus  of  malignant 
edema.  In  their  relation  to  animals  they  also  differ;  for 
instance,  cattle,  while  conspicuously  susceptible  to  symp- 
tomatic anthrax,  are  practically  immune  from  malignant 
edema;  and  while  swine,  dogs,  rabbits,  chickens,  and 
pigeons  are  readily  infected  with  malignant  edema,  they  are 
not,  as  a  rule,  susceptible  to  symptomatic  anthrax.  Horses 


BACTERIUM  WELCHI,  MIGULA  627 

are  affected  only  locally,  and  not  seriously,  by  the  bacillus 
of  symptomatic  anthrax;  but  they  are  conspicuously  sus- 
ceptible to  both  artificial  inoculation  and  natural  infection 
by  the  bacillus  of  malignant  edema. 

The  distribution  of  the  two  organisms  over  the  earth's 
surface  is  also  quite  different.  The  edema  bacillus  is  present 
in  almost  all  soils,  while  the  bacillus  of  symptomatic  anthrax 
appears  to  be  confined  to  certain  localities,  especially  places 
over  which  infected  herds  have  been  pastured. 

A  single  attack  of  symptomatic  anthrax,  if  not  fatal, 
affords  subsequent  protection;  while  infection  with  the 
malignant  edema  bacillus  appears  to  predispose  to  recurrence 
of  the  disease.  (Baumgarten.) 

BACTERIUM  WELCHH,   MIGULA,   1900. 

SYNONYM:     Bacillus  aerogenes  capsulatus,  Welch  and  Nuttall,  1892. 

This  organism  consists  of  straight  or  slightly  curved  rods 
with  rounded  ends,  somewhat  thicker  than  bacterium  an- 
thracis,  varying  in  length  from  3  to  6/z;  sometimes  longer 
chains  or  threads  are  seen.  The  rods  are  surrounded  by  a 
transparent  capsule,  whether  grown  in  artificial  media  or 
obtained  from  animal  bodies.  It  is  a  non-motile,  spore- 
forming  organism,  and  is  strictly  anaerobic  in  character. 
It  stains  with  the  ordinary  aniline  dyes  and  by  the  Gram 
method. 

Under  anaerobic  conditions  the  organism  grows  on  the 
usual  culture  media  at  room  temperature,  and  forms  large 
quantities  of  gas  in  media  containing  carbohydrates.  Gela- 
tin is  not  liquefied.  In  agar-agar  the  colonies  are  usually 
from  1  to  2  millimeters  in  diameter,  but  may  be  as  large  as 
1  centimeter  in  diameter.  They  have  a  grayish-white  color, 


628     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

are  flat,  round  or  irregular  masses,  with  small  hair-like  pro- 
jections from  the  margin.  In  bouillon  there  is  a  diffuse 
clouding  and  marked  white  sediment.  Milk  is  quickly 
coagulated.  On  potato  there  is  a  grayish-white  layer. 

The  organism  grows  more  rapidly  at  30°  to  37°  C.  than 
at  18°  to  20°  C.  Cultures  on  agar-agar  and  bouillon  have 
a  slight  odor  resembling  old  lime. 

Bacterium  Welchii  was  first  described  by  Welch  in  1891, 
and  subsequently  by  Welch  and  Nuttall1  in  the  blood  and 
internal  organs  of  a  patient  with  thoracic  aneurism  opening 
externally.  Autopsy  was  made  eight  hours  after  death  and 
the  vessels  were  found  to  contain  large  numbers  of  gas 
bubbles. 

Injections  of  considerable  quantities  of  cultures  into  the 
circulation  of  rabbits  did  not  kill  the  animals,  but  if  the 
animals  were  killed  after  being  inoculated  and  were  then 
allowed  to  lie  at  room  temperature  for  twenty-four  hours 
the  organs  and  tissues  were  filled  with  gas  bubbles. 

Welch,  Howard,  Hitschman  and  Lilienthal,  Hirschberg, 
and  others  have  shown  that  the  organism  is  frequently 
present  in  the  feces  of  man  and  animals,  as  well  as  in  the 
soil  and  in  dust.  Schattenfroh  and  Grassberger  also  found 
the  organism  in  market  milk. 


BACILLUS   SPOROGENES    (KLEIN),   MIGULA,    1900. 

SYNONYM:    Bacillus  enteritidis  sporogenes,  Klein,  1895. 

Klein  found  this  organism  in  the  intestinal  discharges  of 
infants  and  believed  it  had  some  relation  to  the  acute 
inflammatory  conditions  of  the  intestinal  tract  of  bottle-fed 

«:;  i  Bulletin  Johns  Hopkins  Hospital,  No.  24,  1892. 


THE  SPIROCH&TACEM  629 

infants.  The  organism  is  very  generally  distributed  in 
nature  and  can  be  very  readily  isolated  from  sewage  by 
appropriate  methods.  It  is  an  anaerobic,  spore-forming 
organism,  0.8/JL  in  width,  and  1.6  to  4.8/z  in  length.  It  is 
actively  motile  and  flagella  have  been  demonstrated. 

In  culture  media  containing  carbohydrates  this  organism 
produces  gas  in  large  quantities.  Russell  analyzed  the  gas 
and  found  it  to  be  composed  principally  of  methane.  Milk 
and  other  sugar  media  in  which  the  organism  has  been 
grown  have  a  distinct  odor  of  butyric  acid. 

When  injected  subcutaneously  into  guinea-pigs  this 
organism  causes  most  marked  alterations.  There  is  intense 
inflammation  at  the  point  of  injection  with  edema  and 
necrosis  and  the  surrounding  tissues  are  filled  with  gas. 
The  bacteria  are  distributed  throughout  the  body  of  the 
animal  and  can  be  isolated  in  pure  culture  from  the  blood  of 
the  heart.  All  the  internal  organs  are  intensely  congested. 

THE  SPIROCHJETACE-ffi. 

The  spirochsetacese  may  be  roughly  defined  as  a  family 
of  the  unicellular  non-nucleated,  spiral  organisms,  of  the 
order  spirochetales ,!  which  may  or  may  not  possess  an  undu- 
lating membrane  as  an  extension  from  a  central  filament 
and  which  may  multiply  in  some  instances  by  transverse, 
in  others  by  longitudinal  division. 

This  is  scarcely  the  place  to  discuss  in  detail  the  various 
opinions  that  have  been  expressed  since  the  tune  of  Ehren- 
berg  concerning  the  true  nature  of  this  group  of  micro- 

1  Preliminary  Report,  Committee  on  Classification,  etc.,  Soc.  Am.  Bact., 
Jour.  Bacteriol.,  1917,  No.  5,  vol.  ii.  Studies  in  Classification,  etc.,  by 
R.  E.  Buchanan,  Jour.  Bacteriol.,  1918,  No.  6,  vol.  iii. 


630     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

organisms.  It  will  suffice  to  say  for  our  purposes  that  as 
yet  there  is  no  agreement  as  to  their  exact  status  in  the  world 
of  living  things.  They  appear  to  occupy  a  position  some- 
where between  the  bacteria  on  the  one  hand  and  the  protozoa 
on  the  other. 

A  few  of  them  are  susceptible  of  cultivation  under  arti- 
ficial conditions,  and  on  these  we  possess  at  least  the  begin- 
nings of  an  understanding,  while  for  many  others  which 
cannot  be  cultivated  (or,  at  least,  have  not  been)  we  know 
little  more  than  their  gross  morphological  characteristics. 

A  certain  number  of  them  are  found  in  association  with 
particular  diseases  of  man  and  of  animals  and  are  believed 
to  stand  in  causal  relation  to  such  conditions. 

Many  more  are  found  living  free  in  nature  and  are 
regarded  as  of  no  significance,  insofar  as  disease  production 
is  concerned. 

The  definite  causal  relationship  of  a  few  of  them  to  cer- 
tain diseases  is  now  generally  accepted  as  proved. 

To  the  student  of  etiology  three  genera  in  particular  of 
the  spirochaetacese  are  of  special  interest,  notably:  the  spi- 
ronema,  the  treponema  and  leptospira.1 

Genus  Spironema. — To  this  genus  belong  all  those  species 
or  varieties  that  have  been  seen  in  the  several  manifesta- 
tions of  relapsing  fever  in  man;  such  for  instance  as  spi- 
ronema  carteri,  spironema  obermaieri,  spironema  novyi, 
spironema  duttoni;  also  those  found  in  the  so-called  relaps- 
ing fever  of  fowls,  spironema  gallinarum,  spironema  anserina; 
also  those  found  in  the  mouth,  spironema  buccalis,  spironema 
refringens,  spironema  vincenti  and  a  number  of  others. 

They  are  seen  as  wavy,  irregular  threads  with  pointed 

1  See  Noguchi,  Jour.  Exper.  Med.,  1918,  vol.  xxvii,  p.  575. 


Fig.    106 


Spironema,    Blood,   Relapsing   Fever,  Giemsa  Stain. 

Fig.    1O7 


Treponema   Pallida,  Syphilis,  Silver  Preparation. 


THE  SP1ROCH&TACE&  631 

ends;  varying  in  length  from  8  to  16  /z.  and  in  thickness 
from  0.3  to  0.5  ju.  They  possess  a  delicate  undulating 
membrane,  though  this  is  rarely  seen  in  stained  preparations. 
They  are  motile  but  not  flagellated.  They  multiply  by 
transverse  and  occasionally  by  longitudinal  division. 

When  stained  by  the  Giemsa  method  they  appear  as 
violet  threads  (see  Fig.  106).  When  obtained  in  artificial 
cultivation  they  are  said  (Noguchi)  to  be  soluble  in  10 
per  cent,  bile  salts;  to  be  immobilized  and  ultimately  dis- 
integrated by  10  per  cent,  saponin. 

Genus  Treponema. — To  this  genus  belong  the  species 
treponema  pallada  causing  syphilis  and  treponema  pertenue 
causing  frambesia  tropica  (yaws). 

They  are  seen  in  stained  preparations  and  by  dark  field 
illumination  as  tightly  wound,  short,  rigid  spirals.  They 
vary  in  length  from  6  to  14  /*•  and  in  thickness  of  the  thread 
from  0.2  to  0.3  p.  No  undulating  membrane  is  observed 
(see  Fig.  107). 

Multiply  by  transverse  and  possibly  also  by  longitudinal 
division. 

The  spirals  stain  pink  by  the  Giemsa  method. 

When  obtained  in  culture  (Noguchi)  they  are  dissolved 
by  10  per  cent,  bile  salts  and  more  slowly  by  10  per  cent, 
saponin. 

Genus  Leptospira. — Type  species  leptospira  icerto  hemor- 
rhagise — found  by  Inado  and  Ido  in  infectious  jaundice. 
The  genus  also  includes  leptospira  icteroides  found  by 
Noguchi  in  yellow  fever1  and  described  by  him  as  very 
small,  regular,  rigid  spirals  with  hook-like  prolongations 
at  one  or  both  ends.  They  vary  in  length  from  7  to  14  /z. 

1  See  Jour.  Exper.  Med.,  1919,  vol.  xxx,  p.  13. 


632     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

and  in  thickness  from  0.2  to  0.3  /z.  The  whole  organism 
is  very  flexible  and  has  highly  motile  end-portions.  No 
undulating  membrane'. 

Divide's  transversely. 

Stains  reddish  violet  by  the  Giemsa  method  (see  Fig. 
108).  When  obtained  in  culture  it  is  soluble  in  10  per 
cent,  bile  salts;  insoluble  in  10  per  cent,  saponin. 

Detection  by  Microscopic  Examination. — The  methods 
commonly  employed  in  searching  for  these  organisms  are: 
The  microscopic  examination  of  fresh  unstained  prepara- 
tions by  means  of  the  dark-field  illumination  (see  the 
method),  or  by  the  examination  of  stained  smears.  In 
the  former  method  the  organisms  are  recognized  as  color- 
less, brightly  illuminated,  motile  threads  or  spirals  that 
stand  out  in  striking  contrast  to  the  almost  black  field 
through  which  they  are  moving.  If  the  necessary  equip- 
ment is  available  this  is  by  far  the  quickest  and  most  direct 
method  of  getting  positive  indications. 

By  the  latter  method  the  organisms  are  seen  as. fixed, 
stained  undulating  threads  or  spirals  according  to  their 
nature.  In  this  method  the  examination  is  of  smears  made 
as  follows:  The  smears  are  made  in  the  routine  way  on 
either  clean  cover  slips  or  slides.  They  are  then  allowed 
to  dry  in  the  air,  after  which  they  are  fixed  in  pure  methyl 
alcohol  for  three  or  four  minutes.  They  may  then  be  stained 
by  the  Giemsa  method  or  by  some  one  or  another  of  the 
silver  impregnation,  methods. 


STAINING   SOLUTION. 

Azure  II  (eosin) 0.30  grams 

Azure  II  .  ...       0.08       u 


Fig.  108 


Leptospira  leteroides.      Blood  of  Experiment  Guinea-pig. 
(After  Noguehi) 


THE  SPIROCHMTACEM  633 

Mix  with  25  c.c.  of  pure  anhydrous  glycerin  at  60°  C. 
When  dissolved  add  25  c.c.  of  pure  methyl  alcohol,  also  at 
60°  C.,  allow  mixture  to  stand  over-night,  then  filter. 
To  stain  the  smears  take  from  the  above  "stock"  solution 
1  c.c.  and  mix  with  10  or  12  c.c.  of  1 : 1000  potassium  car- 
bonate in  distilled  water.  Pour  this  diluted  stain  over 
the  smears  and  allow  to  stand  for  from  a  quarter  to  half 
an  hour.  If  the  smear  be  very  thick,  exposure  to  the  stain 
should  be  longer.  Wash  repeatedly  in  clean  water  until  the 
smear  has  in  general  a  pink  tinge.  This  clearing  up  may  be 
hastened  in  thick  smears  by  immersion  for  an  instant  in 
methyl  alcohol,  followed  by  repeated  washings  in  water. 
When  dried,  the  preparations  are  examined  in  the  way 
common  to  microscopic  examination  of  bacterial  prepara- 
tions. 

Silver  Impregnation.  Stern's  Method. — Prepare  smear; 
dry  in  incubator  at  37°  to  38°  C.  for  several  hours  (do  not 
heat  over  flame). 

Immerse  in  10  per  cent,  silver  nitrate  solution  and  expose 
to  diffuse  day-light  (not  direct  sun-light)  for  from  several 
hours  to  several  days.  When  the  preparation  is  of  a  deep 
brown  color  and  shows  a  metallic  sheen,  wash  thoroughly 
in  clean  water,  dry  and  mount  for  examination. 

The  organisms  are  seen  as  black  threads  or  spirals  in  a 
brown  field. 

India  Milk  Method. — Mix  a  drop  of  the  suspected  blood 
or  exudate  with  a  drop  of  India  milk  on  one  end  of  a  slide. 
With  the  edge  of  another  slide  draw  this  quickly  down  the 
length  of  the  slide  so  as  to  make  an  even  tolerably  thin  film. 
Allow  to  dry.  Examine  by  the  usual  method.  Any  organ- 
isms present  will  appear  as  colorless  objects  in  an  almost 
black  field.  The  picture  is  that  of  a  photographic  negative. 


634     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

Levaditis  Method  for  Tissues. — Fix  bits  of  suspected  tissue, 
not  over  2  m.m.  thick,  in  10  per  cent,  formalin  for  one  or 
two  days.  Wash  out  in  95  per  cent,  alcohol  for  eighteen  to 
twenty  hours.  Wash  thoroughly  in  distilled  water  until  the 
tissues  no  longer  float. 

Impregnate  in: 

Nitrate  of  silver 1  gram 

Pyridin 10  grams 

Distilled  water 100  c.c. 

Made  up  fresh. 

Allow  to  stand  at  room  temperature  for  two  or  three 
hours,  followed  by  50°  C.  for  from  four  to  six  hours.  Wash 
rapidly  in  10  per  cent,  pyridin,  after  which  reduce  the  silver 
,by  immersing  the  tissue  for  several  hours  in: 

Pyrogallic  acid    ...;.".,....  4  grams 

Acetone 10      " 

Pyridin 15      " 

Distilled  water 100  c.c. 

Made  up  fresh. 

Harden  in  alcohol.  Embed  in  paraffin  and  cut  sections 
about  5  /*.  thick;  mount  in  balsam.  The  spirochetes  appear 
in  tissues  so  treated  as  intensely  black  objects. 

Cultivation.1  —  Certain  of  the  spirochsetacese  lend  them- 
selves to  artificial  cultivation;  others  have  eluded  all  such 
efforts.  There  are  not  as  yet  any  standard  methods  of 
artificial  cultivation  such  as  are  used  in  the  routine  study 
of  "bacteria.  Special  methods  have  been  devised  by  a 
number  of  investigators  and  all  have  met  with  more  or  less 
success.  Practically  all  such  methods  depend  for  success 
upon  certain  fundamental  requirements: 

The  culture  fluids  must  consist  essentially  of  sterile  animal 
juices  or  exudates;  must  contain  bits  of  fresh  animal  tissue; 

1  See  Noguchi,  Jour.  Exper.  Med.,  1918,  vol.  xxvii,  p.  593. 


THE  SPIROCHMTACE&  635 

must  in  some  instances  present  strict,  in  others  only  partial 
anaerobic  conditions. 

The  preparation  of  such  culture  media  is  done  under  strict 
aseptic  conditions  as  the  heating  of  the  media  for  purposes 
of  final  sterilization  robs  them  of  their  usefulness. 

Noguchi,  whose  investigations  in  this  field  have  con- 
tributed so  much  to  our  knowledge  on  the  subject,  finds 
that  in  general  a  medium  made  up  of  about  12  to  15  c.c. 
of  sterile  ascitic  or  hydrocele  fluid  to  which  is  added  a  few 
drops  of  citrated  blood  and  a  bit  (about  the  size  of  a  bean) 
of  fresh  rabbit  kidney,  serves  very  well  for  the  cultivation 
of  most  blood  spirochetes.  He  has  also  had  success  with 
the  following  mixture: 

Rabbit  serum ;  .    1.5  parts 

Ringer's  solution1     .      . 4.5      " 

Citrate  plasma 1.0      " 

If  it  be  desirable  to  stiffen  the  medium,  and  certain 
spirochetes  develop  better  in  such  than  in  fluid  media,  sterile 
agar  (free  of  peptone  and  sugar)  in  the  proportion  of  2  per 
cent,  may  be  added. 

For  those  species  requiring  partial  anaerobic  conditions 
a  little  sterilized  paraffin  oil  may  be  run  over  the  surface. 
For  those  requiring  strict  anaerobic  conditions  tubes  should 
be  kept  in  an  oxygen-free  atmosphere  (see  anaerobic 
methods) . 

The  citrated  blood  in  the  above  mixture  may  be  drawn 
under  aseptic  conditions  from  the  animal  or  person  whose 

1  Ringer's  solution: 

Sodium  chloride 10.0  grams 

Potassium  chloride 0.2       " 

Calcium  chloride 0.2       " 

Sodium  bicarbonate 0.1  .    " 

Glucose 1.0       " 

Water                                                                       .  1000.0  c.c. 


636     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

blood  contains  the  organism  to  be  cultivated.  The  value 
of  this  medium  is  destroyed  by  the  addition  of  either  bouillon 
or  sugar;  and  the  bit  of  kidney  should  be  taken  from  a 
freshly  killed  animal.  The  ascitic  fluid  should  be  free 
from  bile  and  when  placed  in  the  test-tube  should  permit 
of  the  formation  of  a  loose  fibrin  meshwork.  The  culti- 
vation should  be  conducted  at  37°  to  38°  C.  The  growth 
of  the  organisms  in  these  media  causes  no  appreciable 
change  in  appearance.  Multiplication  of  the  organisms  is 
noted  after  two  or  three  days  at  body  temperature.  Sub- 
cultures should  be  made  between  the  fourth  and  ninth 
days.  After  about  ten  days  the  multiplication  of  the 
organisms  ceases.  After  several  generations  of  artificial 
cultivation  the  spirochetes  gradually  lose  the  power  to 
infect  susceptible  animals. 

In  the  various  manifestations  of  relapsing  fever  the 
organisms  are  found  in  the  circulating  blood;  in  syphilis 
they  are  found  in  the  diseased  tissues  and  in  the  juices 
squeezed  from  the  primary  sore  and  from  other  superficial 
lesions;  in  yellow  fever  the  leptospira  icteroides  is  found 
in  the  blood. 


CHAPTER  XXIX. 

Bacteriological  Study  of  Water — Methods  Employed — Precautions  to  be 
Observed — Apparatus  Employed,  and  Methods  of  Using  It — Methods 
of  Investigating  Air  and  Soil — Bacteriological  Study  of  Milk — Methods 
Employed. 


BACTERIOLOGICAL   STUDY   OF  WATER. 

THE  conditions  that  favor  epidemic  outbreaks  of  typhoid 
fever,  Asiatic  cholera,  and  other  maladies  of  which  these 
may  be  taken  as  types,  have  served  as  a  subject  for  dis- 
cussion by  sanitarians  for  a  long  time. 

Of  the  opinions  that  have  'been  advanced  in  explanation 
of  the  existence  and  dissemination  of  these  diseases,  two 
should  be  considered:  one,  the  ground-water  doctrine  of 
von  Pettenkofer,  because  of  its  historic  interest;  the  other, 
the  belief  that  the  diseases  are  disseminated  by  specifically 
polluted  waters,  bacause  it  is  the  view  now  prevalent  among 
modern  sanitarians. 

The  advocates  of  the  "ground-water"  view  explained 
the  occurrence  of  these  diseases  in  epidemic  form  through 
alterations  in  the  soil  resulting  from  fluctuations  in  the 
level  of  the  soil- water;  and  assigned  to  drinking-water 
either  a  very  insignificant  role,  or  ignored  it  entirely.  On 
the  other  hand,  those  who  have  been  instrumental  in 
developing  the  drinking-water  hypothesis  claim  that  altera- 
tions in  the  soil  play  little  or  no  part  in  favoring  epidemic 
outbreaks;  but  that,  as  a  rule,  they  appear  as  a  result  of 
direct  infection,  through  the  use  of  waters  contaminated 

(637) 


638     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

with  materials  containing  the  specific  organisms  known  to 
cause  such  diseases. 

As  a  result  of  evidence  now  known  to  everyone  it  is  the 
general  belief  that  polluted  water  is  primarily  the  under- 
lying cause  of  most  widespread  epidemics  of  intestinal 
infections  and  this  too,  very  often,  when  the  state  of  the 
soil-water,  in  the  light  of  the  "ground-water"  hypothesis,  is 
just  the  reverse  of  what  it  should  be  in  order  to  render  it 
answerable  for  them.  It  is  manifest,  therefore,  that  the 
careful  bacteriological  study  of  water  intended  for  domestic 
use  is  of  the  greatest  importance,  and  should  be  a  routine 
procedure  in  all  communities  receiving  their  water-supply 
from  sources  liable  to  pollution. 

The  object  aimed  at  in  such  investigations  should  be  to 
determine  the  number  and  kind  of  bacteria  constantly 
present  in  the  water — for  all  waters,  except  deep  ground- 
water,  contain  bacteria;  if  sudden  fluctuations  in  the 
number  and  kind  of  bacteria  occur  in  these  waters,  and  if 
so,  to  what  they  are  due;  and  finally,  and  most  important, 
whether  the  water  contains  constantly,  or  at  irregular 
periods,  bacteria  that  can  be  traced  to  human  excrement, 
not  of  necessity  pathogenic  varieties,  but  bacteria  that  are 
known  to  be  present  normally  in  the  intestinal  canal.  For 
if  conditions  are  continuously  favorable  to  pollution  of  the 
water  by  the  normal  constituents  of  the  intestinal  canal, 
the  same  conditions  would  allow  of  the  occasional  pollution 
of  such  water  by  infective  matters  from  the  bowels  of  persons 
suffering  from  specific  disease  of  the  intestines. 

In  considering  water  from  a  bacteriological  standpoint  it 
must  always  be  borne  in  mind  that  comparisons  with  fixed 
standards  are  not  of  much  value,  for  just  as  normal  waters 
from  different  sources  are  seen  to  present  variations  in  their 


BACTERIOLOGICAL  STUDY  OF  WATER  639 

chemical  composition,  without  necessarily  being  unfit  for  use, 
so  may  the  relative  number  and  variety  of  species  of  bacteria 
in  water  from  one  source  be  always  greater  or  smaller  than 
in  that  from  another,  and  yet  no  difference  may  be  seen 
to  result  from  their  employment.  For  this  reason  systematic 
study  of  any  water,  from  this  point  of  view,  should  begin 
with  the  establishment  of  what  may  be  called  its  normal 
mean  number  of  bacteria,  as  well  as  the  character  of  the 
prevailing  species;  and  in  order  to  do  this  the  investigations 
must  cover  a  long  period  of  time  through  all  the  seasonal 
variations  of  weather.  From  data  obtained  in.  this  way  it 
may  be  possible  without  analysis  to  predict  approximately 
at  any  season  the  bacteriological  condition  of  the  water 
studied.  Marked  deviations  from  these  "means,"  either 
in  the  quantity  or  quality  of  the  organisms  present,  can 
then  be  considered  as  indicative  of  the  existence  of  some 
unusual,  disturbing  element,  the  nature  of  which  should  be 
investigated.  It  is  impossible  to  formulate  an  opinion  of 
much  value  from  either  a  single  chemical  or  bacteriological 
analysis  of  a  water,  or  from  both  together  in  many  cases; 
for  the  results  thus  obtained  indicate  only  the  condition 
of  the  water  at  the  time  the  sample  was  procured,  and  give 
no  indication  as  to  whether  it  differed  at  that  time  from  its 
usual  condition,  or  from  the  normal  condition  of  the  waters 
of  the  immediate  neighborhood. 

The  interpretation  of  the  results  of  both  chemical  and 
bacteriological  analyses  of  a  sample  of  water  acquires  its 
full  value  only  through  comparison,  either  with  "means" 
that  have  been  determined  for  this  water,  or  with  the  results 
of  simultaneous  analyses  of  a  number  of  samples  from  other 
sources  of  supply  of  the  locality. 

The  aid  of  the  bacteriologist  is  frequently  sought  in  con- 


640     APPLICATION  OF  METHODS  OF   BACTERIOLOGY 

nection  with  investigations  of  waters  that  are  supposed  to 
be  concerned  in  the  production  of  disease,  particularly 
typhoid  fever,  either  in  isolated  cases  or  in  widespread 
epidemic  outbreaks,  and  in  these  cases  both  the  bacteriolo- 
gist and  the  person  employing  his  services  are  cautioned 
against  being  too  sanguine  of  positive  results,  for  in  the 
vast  majority  of  instances  reliable  bacteriologists  fail  to 
detect  in  these  waters  the  bacillus  that  is  the  cause  of 
typhoid  fever. 

Failure  to  find  the  organism  of  typhoid  fever  in  water  by 
the  usual  methods  of  analysis  does  not  by  any  means  prove 
that  it  is  not  present  or  has  not  been  present.  The  means 
ordinarily  employed  in  the  work  admit  of  such  a  very  small 
volume  of  water  being  used  in  the  test  that  we  can  readily 
understand  how  typhoid  bacilli  might  be  present  in  moderate 
numbers  and  yet  none  be  included  in  the  drop  or  two  of  the 
water  taken  for  study.  The  conditions  are  not  those  of  a 
solution,  each  drop  of  which  contains  exactly  as  much  of 
the  dissolved  material  as  do  all  other  drops  of  equal  volume; 
but  are  rather  those  of  a  suspension,  in  every  drop  or  volume 
of  which  the  number  of  suspended  particles  is  liable  to  the 
greatest  degree  of  variation.  Furthermore,  there  are  other 
reasons  that  would,  a  priori,  preclude  our  expecting  to  find 
the  typhoid  bacilli  in  water  in  which  we  may  have  reason 
to  believe  they  had  been  deposited,  because  attention  is 
not  usually  directed  to  the  water  until  the  disease  has  become 
conspicuous,  usually  in  from  two  to  four  weeks  after  the 
pollution  probably  occurred.  These  intervals  of  time  are 
ordinarily  sufficient  for  the  delicate,  non-resistant  bacillus 
of  typhoid  fever  to  succumb  to  the  unfavorable  conditions 
under  which  it  finds  itself  in  water.  By  unfavorable  con- 
ditions are  meant  the  absence  of  suitable  nutrition;  un- 


BACTERIOLOGICAL  STUDY  OF   WATER  641 

favorable  temperature;  probably  the  antagonistic  influence 
of  more  hardy  saprophytic  bacteria,  particularly  the  so- 
called  "water-bacteria,"  and  of  more  highly  organized 
water-plants;  the  effect  of  precipitation  and  of  sedimenta- 
tion; and,  of  great  importance,  the  disinfecting  action  of 
direct  sunlight. 

Though  the  positive  demonstration  of  typhoid  bacilli  in 
drinking-water  by  bacteriological  methods  is  of  extreme 
rarity,  it  must  not  be  concluded  that  bacteriological  analyses 
of  suspicious  waters  shed  no  light  upon  the  existence  of 
pollution  and  the  suitability  or  non-suitability  of  the  water 
for  drinking-purposes. 

In  the  normal  intestinal  tract  of  human  beings  and 
domestic  animals,  as  well  as  associated  with  the  specific 
disease-producing  bacillus  in  the  intestines  of  typhoid- 
fever  patients,  is  an  organism  that  is  frequently  found  in 
polluted  drinking-waters,  and  whose  presence  is  indicative 
of  pollution  by  either  normal  or  diseased  intestinal  con- 
tents; and  though  efforts  may  result  in  failure  to  detect 
the  specific  bacillus  of  typhoid-fever,  the  finding  of  the 
other  organism,  bacillus  coli,  justifies  one  in  concluding 
that  the  water  under  consideration  has  been  polluted  by 
intestinal  evacuations  from  either  human  beings  or  animals. 
Waters  so  exposed  as  to  be  liable  to  such  pollution  should 
never  be  considered  as  other  than  a  continuous  source  of 
danger  to  those  using  them. 

Another  point  to  be  remembered  is  in  connection  with 
chlorine  as  an  indicator  of  contamination  by  human  excre- 
ment. It  is  commonly  taught  that  an  excessive  amount 
of  chlorine  in  water  points  to  contamination  by  human 
excreta.  This  may  or  may  not  be  true,  according  to  cir- 
cumstances. A  high  proportion  of  this  element  in  a  sample 
41 


642     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

of  water  from  a  locality,  the  surrounding  waters  of  which 
are  poor  in  chlorine,  is  unquestionably  a  suspicious  indica- 
tion; but  in  a  district  close  to  the  sea  or  near  salt-deposits, 
for  instance,  where  the  proportion  of  chlorine  (as  chlorides) 
in  the  water  is  generally  high,  the  value  of  the  indications 
thus  afforded  is  very  much  diminished  unless  the  amount 
found  in  the  sample  under  examination  greatly  exceeds  the 
normal  "mean,"  previously  determined,  for  the  amount  of 
chlorine  in  the  waters  of  the  neighborhood. 

A  striking  example  of  the  latter  condition  occurred  in 
the  experience  of  the  writer  while  inspecting  a  group  of 
water-supplies  on  the  east  coast  of  Florida.  In  each  in- 
stance the  water  was  obtained  from  properly  protected 
artesian  wells,  ranging  from  200  to  400  feet  deep,  and  located 
within  a  few  hundred  yards  of  the  sea.  The  first  sample 
subjected  to  chemical  analysis  revealed  such  an  unusually 
high  proportion  of  chlorine  that,  had  this  sample  alone 
been  considered,  the  opinion  that  it  was  polluted  by  human 
excreta  might  have  been  advanced.  To  prevent  such  an 
error  samples  of  water  from  a  number  of  wells  in  the  neigh- 
borhood were  examined,  and  they  were  all  found  to  contain 
from  ten  to  twelve  times  the  amount  of  chlorine  that  ordi- 
narily appears  in  inland  waters,  the  excess  being  evidently 
due  to  leakage  through  the  soil  into  the  wells  of  water  from 
the  sea.  In  short,  the  presence  of  an  excess  of  chlorine  in 
water,  while  often  indicating  pollution  from  human  evacua- 
tions, may  nevertheless,  sometimes  arise  from  other  sources; 
but  the  presence  in  water  of  bacteria  normally  found  in 
the  intestinal  canal  can  manifestly  admit  of  but  one  inter- 
pretation, viz.,  that  fecal  matters  from  either  man  or 
animals  have  at  some  time  been  deposited  in  this  water, 
and  that  while  no  specific  disease-producing  organisms  may 


BACTERIOLOGICAL  STUDY  OF  WATER  643 

accompany  them,  still  waters  in  which  such  pollutions  are 
possible  are  also  open  to  other  dangerous  pollutions,  and 
must  be  regarded  as  a  constant  menace  to  the  health  of 
those  who  use  them  for  domestic  purposes. 

A  sudden  variation  from  the  normal,  mean  number  of 
bacteria,  or  from  the  normal  chemical  composition  of  a 
water,  calls  at  once  for  a  thorough  inspection  of  the  supply, 
while  at  the  same  time  the  organisms  present  are  to  be  sub- 
jected to  the  most  careful  study.  In  many  instances,  even 
after  the  most  thorough  bacteriological  and  chemical  study 
of  a  suspicious  water,  one  is  forced  to  admit  that  informa- 
tion of  but  limited  usefulness  has  been  obtained  through 
the  employment  of  such  analytical  methods.  In  these 
cases  too  much  stress  cannot  be  laid  upon  the  importance 
of  a  systematic  inspection  of  the  supply,  and  its  relation 
to  sources  of  pollution.  Optical  evidence  of  more  or  less 
dangerous  contamination  may  often  be  obtained  when 
laboratory  methods  fail  to  detect  them.  The  reasons  for 
such  failure,  in  addition  to  those  already  given,  are  obvious — 
the  polluting  matters  are  often  so  diluted  by  the  large 
mass  of  water  into  which  they  find  their  way  as  to  be  beyond 
recognition  by  the  tests  usually  employed  in  such  work, 
and  still  be  present  in  amounts  sufficient  to  originate 
disease. 

The  Qualitative  Bacteriological  Analysis  of  Water. — The 
qualitative  bacteriological  analysis  of  water  entails  much 
labor,  as  it  requires  not  only  that  all  the  different  species 
of  organisms  found  in  the  water  should  be  isolated,  but 
that  each  representative  should  be  subjected  to  systematic 
study,  and  its  pathogenic  or  non-pathogenic  properties 
determined. 

For  this  purpose  a  knowledge  of  the  methods  for  the 


644     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

isolation  of  individual  species  which  have  been  described 
already,  and  of  the  means  of  studying  these  species  when 
isolated,  is  indispensable. 

For  this  analysis  certain  precautions  essential  to  accuracy 
are  always  to  be  observed. 

The  sample  is  to  be  collected  under  the  most  rigid  pre- 
cautions that  will  exclude  organisms  from  sources  other 
than  that  under  consideration.  If  drawn  from  a  spigot, 
it  should  never  be  collected  until  the  water  has  been  flowing 
for  fifteen  to  twenty  minutes  in  a  full  stream.  If  obtained 
from  a  stream  or  a  spring,  it  should  be  collected,  not  from 
the  surface,  but  rather  from  about  one  foot  beneath  the 
surface. 

It  should  always  be  collected  in  vessels  which  have  pre- 
viously been  thoroughly  freed  from  all  dirt  and  organic 
particles,  and  then  sterilized;  and  the  plates  should  be 
made  as  quickly  as  possible  after  collecting  the  sample. 

When  circumstances  permit,  all  water  analyses  should  be 
made  on  the  spot  where  the  sample  is  taken,  as  it  is  known 
that  during  transportation,  unless  the  samples  are  kept 
packed  in  ice,  a  multiplication  of  the  organisms  contained 
in  it  always  occurs. 

For  the  purpose  of  qualitative  analysis  it  is  necessary 
that  a  small  portion  of  the  water — one,  two,  three,  five 
drops — should  first  be  employed  for  making  the  plates. 
In  this  way  one  can  form  an  idea  as  to  the  approximate 
number  of  organisms  in  the  water,  and  can,  in  consequence, 
determine  the  amount  of  water  best  suited  for  the  plates. 
Duplicate  plates  are  always  to  be  made — one  set  upon 
agar-agar,  which  are  to  be  kept  in  the  incubator  at  body- 
temperature,  and  one  set  upon  gelatin,  to  be  kept  at  from 
18°  to  20°  C. 


BACTERIOLOGICAL  STUDY  OF  WATER  645 

As  soon  as  colonies  have  developed  the  plates  are  to  be 
carefully  compared  and  studied.  It  is  to  be  noted  if  any 
difference  in  the  appearance  of  the  organisms  on  correspond- 
ing plates  exists,  and  if  so,  to  what  it  is  due.  It  is  to  be 
particularly  noted  which  plates  contain  the  greater  number 
of  colonies,  those  kept  at  the  higher  or  those  at  the  lower 
temperature.  In  this  way  the  temperature  best  suited 
for  the  growth  of  the  majority  of  these,  organisms  may  be 
determined.  As  a  rule,  the  greater  number  of  colonies 
appear  upon  the  gelatin  plates  kept  at  18°  to  20°  C.;  and 
from  this  it  would  seem  that  many  of  the  normal  water- 
bacteria  do  not  find  the  higher  temperature  so  favorable 
to  their  development  as  do  the  organisms  not  naturally 
present  in  water,  particularly  the  pathogenic  varieties. 
From  these  plates  the  different  species  are  to  be  isolated 
in  pure  culture,  the  morphological  and  cultural  character- 
istics determined,  and  finally,  by  tests  upon  animals,  it  is 
to  be  decided  if  any  of  them  possess  disease-producing 
properties. 

NOTE. — What  use  should  be  made  of  this  observation 
in  examining  water  for  the  presence  of  pathogenic  bacteria? 

The  waters  most  frequently  studied  from  the  qualitative 
bacteriological  standpoint  are  those  suspected  of  containing 
specific  pathogenic  bacteria — i.  e.}  waters  polluted  with 
sewage  and  with  human  excreta  that  are  believed  to  be  the 
source  of  infection  of  typhoid  fever,  or,  less  frequently, 
of  Asiatic  cholera.  In  the  investigations  of  such  water 
there  are  several  points  of  which  we  should  never  lose  sight, 
viz.,  unless  the  water  is  under  continuous  study  there  is 
only  a  chance  of  detecting  the  specific  pathogenic  species, 
for,  as  a  rule,  the  dangerous  pollution  occurs  either  but 


646     APPLICATION  OF  METHODS  OF   BACTERIOLOGY 

once  or  is  intermittent,  so  that  even  in  the  case  of  exposed 
streams  there  are  periods  when  no  specifically  dangerous 
contamination  may  be  in  operation.  As  stated  above 
attention  is  commonly  called  to  the  water  when  the  disease, 
presumably  caused  by  its  use,  is  fully  developed,  and  this 
is  often  days  or  weeks  after  the  pollution  of  the  stream  really 
may  have  occurred.  By  an  analysis  made  at  this  time  one 
could  scarcely  hope  to  detect  the  specific  organisms  that 
had  caused  the  disease,  especially  in  water  from  flowing 
streams.  The  organisms  sought  for  may  have  been  present 
in  the  water  and  may  have  infected  the  users,  and  yet  have 
disappeared  by  the  time  the  sample  taken  for  analysis  was 
collected. 

When  present  in  polluted  waters  pathogenic  bacteria  are 
always  vastly  in  the  minority.  Were  they  constantly 
present  in  large  numbers  infection  among  the  users  of  such 
waters  would  be  more  frequent  and  more  widespread  than 
is  commonly  the  case.  They  may  be  present  in  a  water- 
supply  in  small  numbers;  they  may  even  be  in  the  sample 
supplied  for  analysis,  and  yet  escape  detection  if  only  the 
ordinary  direct  plate  method  of  isolation  be  used. 

From  these  considerations  it  is  obvious  that  before 
attempts  are  made  to  isolate  the  various  species  .directly 
from  a  suspicious  sample  of  water  it  is  advisable  to  subject 
it  to  some  method  of  treatment  that  will  aid  in  separating 
the  few  specific  pathogenic  from  the  numerous  common 
saprophytic  species.  For  this  purpose  numerous  so-called 
methods  of  "enrichment"  have  been  devised.  The  most 
useful  of  these  aim  to  favor  the  rapid  multiplication  of 
pathogenic  forms  that  may  be  present  and  to  suppress  or 
check  the  growth  of  the  ordinary  water  saprophytes. 

Attention  has  been  called  to  the  fact  that  when  exposed 


BACTERIOLOGICAL  STUDY  OF  WATER  647 

to  the  body-temperature  many  of  the  ordinary  water- 
bacteria  develop  only  slowly  or  not  at  all,  while  under 
similar  circumstances  the  disease-producing  species  develop 
most  luxuriantly.  Advantage  has  been  taken  of  this  obser- 
vation in  devising  methods  for  this  particular  work,  of 
which  some  of  the  following  will  prove  serviceable: 

Collect  in  a  sterilized  flask  a  sample  of  about  100  c.c. 
of  the  water  to  be  tested,  and  add  to  this  about  25  c.c.  of 
sterilized  bouillon  of  four  times  the  usual  strength.  This  is 
then  placed  in  the  incubator  at  37°  to  38°  C.,  for  thirty-six 
to  forty-eight  hours,  after  which  plates  are  to  be  made  from 
it  in  the^usual  way;  the  results  will  often  be  a  pure  culture 
of  some  single  organism,  either  one  of  the  intestinal  variety 
or  a  closely  allied  species.  By  a  method  analogous  to  the 
latter  the  spirillum  of  Asiatic  cholera  has  been  isolated  from 
water  (see  article  on  that  organism) ;  and  by  taking  advan- 
tage of  the  effect  of  elevated  temperature  upon  the  bacteria 
of  water  Vaughan  has  succeeded  in  isolating  from  suspicious 
waters  a  group  of  organisms  very  closely  allied  to  the  bacillus 
of  typhoid  fever. 

Theobald  Smith  has  suggested  a  method  by  which  it  is 
easily  possible  to  isolate,  from  waters  in  which  they  are 
present,  certain  organisms  that  are  of  the  utmost  impor- 
tance in  influencing  our  judgment  upon  the  fitness  of  the 
water  for  domestic  use.  By  the  addition  of  small  quantities 
— one,  two,  or  three  drops — of  the  suspicious  water  to 
fermentation-tubes  (see  article  on  Fermentation-tube)  con- 
taining bouillon  to  which  2  per  cent,  of  glucose  has  been 
added,  and  keeping  them  at  the  temperature  of  the  body 
(37°  to  38°  C.),  the  growth  of  intestinal  bacteria  that  may 
be  present  in  the  water  is  favored,  while  that  of  the  water- 
organisms  is  not;  in  consequence,  after  from  thirty-six  to 


648     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

forty-eight  hours  the  fermentation  characteristics  of  most 
of  these  organisms  is  evidenced  by  the  accumulation  of  gas 
in  the  closed  end  of  the  tube.  From  these  tubes  the  growing 
bacteria  can  then  be  easily  isolated  by  the  plate  method, 
and  intestinal  bacteria  will  not  infrequently  be  found  present. 
For  the  isolation  of  the  typhoid  bacillus,  especially  from 
water,  a  host  of  other  methods  have  been  devised.  Some  of 
these  aim,  through  the  addition  of  special  chemical  reagents 
to  the  media,  to  retard  the  development  of  ordinary  sapro- 
phytes without  interrupting  the  growth  of  the  colon  and 
the  typhoid  bacillus.  Most  of  these  methods  have  proved 
disappointing.  One  of  them,  that  of  Parietti,  still  finds 
favor  in  the  hands  of  some.  It  consists  in  adding  to  the 
culture  media  to  be  used  in  the  test  varying  amounts  of 
the  following  mixture : 

Phenol 5  grams 

Hydrochloric  acid 4  grams 

Distilled  water 100  c.c. 

Of  this  solution  0.1,  0.2,  and  0.3  c.c.  are  added  respectively 
to  each  of  three  tubes  containing  10  c.c.  of  nutritive  bouillon. 
Several  such  sets  of  tubes  are  to  be  made.  To  each  are 
then  added  from  1  to  3  c.c.  of  the  water,  and  they  are  placed 
in  the  incubator  at  body-temperature.  It  is  said  that 
whatever  development  occurs  consists  only  of  the  typhoid 
or  colon  bacillus,  or  both,  if  they  were  present  in  the  original 
sample.  They  may  then  be  isolated  and  separated  by  the 
usual  plate  method,  or,  better  still,  through  the  application 
of  the  methods  of  v.  Drigalski  and  Conradi,  of  Ficker,  or 
of  Hoffmann  and  Ficker,  or  several  of  these  methods  in 
conjunction,  detailed  in  the  chapter  on  bacillus  typhosus. 
Personally  we  have  not  had  much  success  with  the  Parietti 
method.  The  typhoid  bacillus  has  been  isolated  from  water 


BACTERIOLOGICAL  STUDY  OF  WATER  649 

by  passing  very  large  quantities  of  water  through  an  ordinary 
Pasteur  or  Berkefeld  filter,  brushing  off  the  matters  collected 
on  the  filter  into  a  sterilized  vessel  and  examining  this  by 
plate  methods. 

It  has  occurred  to  us  that  possibly  the  employment  of 
chemical  coagulants,  such  as  alum  and  iron,  might  prove 
serviceable  for  this  purpose.  Their  action  would  be  to 
mechanically  drag  down,  in  precipitating  as  hydroxides, 
the  suspended  bacteria  contained  in  the  fluid.  This  preci- 
pitate could  then  be  examined  bacteriologically,  *  instead 
of  the  water,  and  the  recent  experiments  of  Ficker  (loc.  tit.) 
appear  to  demonstrate'  the  value  of  such  a  procedure. 

The  difficulties  in  this  field  of  work  are  obviously  due  to 
the  suspension  of  a  very  small  number  of  the  disease-pro- 
ducing organisms  sought  for  in  large  volumes  of  fluid,  and 
the  association  with  them  of  large  numbers  of  other  species 
that  offer  a  very  great  obstacle  to  the  successful  search  for 
the  pathogenic  varieties. 

If  by  either  of  the  above  procedures  bacilli  that  bear 
any  resemblance  to  bacillus  typhosus  be  isolated,  recourse 
must  then  be  had  to  all  the  differential  tests  detailed  in  the 
chapter  on  that  organism. 

The  Quantitative  Estimation  of  Bacteria  in  Water. — Quan- 
titative analysis  requires  more  care  in  the  measurement  of 
the  exact  volume  of  water  employed,  for  the  results  are  to 
be  expressed  in  terms  of  the  number  of  individual  organisms 
to  a  definite  volume.  The  necessity  for  making  the  plates 
at  the  place  at  which  the  sample  is  collected  is  to  be  particu- 
larly accentuated  in  this  analysis,  for  multiplication  of  the 
organisms  during  transit  is  so  great  that  the  results  of 
analyses  made  after  the  water  has  been  in  a  vessel  for  a 
day  or  two  are  often  very  different  from  those  that  would 
have  been  obtained  on  the  spot. 


650     APPLICATION  OF  METHODS   OF   BACTERIOLOGY 

NOTE. — Inoculate  a  tube  containing  about  ten  cubic 
centimeters  of  sterilized  distilled  or  tap  water  with  a  very 
small  quantity  of  a  solid  culture  of  some  one  of  the  organ- 
isms with  which  you  have  been  working,  taking  care  that 
none  of  the  culture  medium  is  introduced  into  the  water- 
tube  and  that  the  bacteria  are  evenly  distributed  through 
it.  Make  plates  at  once  from  this  tube,  and  on  each  suc- 
ceeding day  determine  by  counts  whether  there  is  an  increase 
or  diminution  in  the  number  of  organisms — i.  e.,  if  they  are 
growing  or  dying.  Represent  the  results  graphically,  and 
it  will  be  noticed  that  in  many  cases  there  is  during  the 
first  three  or  four  days  a  multiplication,  after  which  there 
is  a  rapid  diminution;  and,  if  the  organism  does  not  form 
spores,  usually  death  in  from  ten  to  twelve  days.  This  is 
not  true  for  all  organisms,  but  does  hold  for  many. 

Where  it  is  not  convenient,  however,  to  make  the  analysis 
on  the  spot,  the  sample  of  water  should  be  taken  and  packed 
in  ice  and  kept  on  ice  until  the  plates  can  be  made  from  it, 
which  should  in  all  cases  be  as  soon  after  its  collection  as 
possible. 

For  the  collection  of  samples  from  the  deeper  portions 
of  streams,  lakes,  etc.,  a  number  of  convenient  devices  have 
been  made.  A  very  satisfactory  apparatus  has  been  made 
for  me  by  Messrs.  Charles  Lentz  &  Sons,  of  Philadelphia. 
It  consists  of  a  metal  frame-work,  in  which  is  encased  a 
bottle  provided  with  a  ground-glass  stopper.  To  the  stopper 
a  spring  clamp  is  attached,  and  this  in  turn  is  operated  by 
a  string,  so  that  when  the  weighted  apparatus  is  allowed  to 
sink  into  the  stream  the  stopper  may  be  removed  from  the 
bottle  at  any  depth  by  simply  pulling  upon  the  string. 
When  the  bottle  is  filled  with  water  the  stopper  is  allowed 
to  spring  back  into  position  by  releasing  the  string.  The 


BACTERIOLOGICAL  STUDY  OF  WATER 


651 


FIG.  109 


whole  apparatus  (depicted  in  Fig.  109)  is  provided  with  a 
weight  that  insures  its  sinking,  and  a  heavy  cord  by  which 
it  may  be  lowered  and  raised.  It  should  be  sterilized  before 
using.  After  collecting  the  sample  the 
bottle  should  be  wiped  dry  with  a 
sterilized  towel.  Before  removing  the 
stopper  the  mouth  of  the  bottle  should  be 
rinsed  with  alcohol  and  heated  with  a  gas- 
flame,  to  prevent  contamination  of  its 
contents  by  matters  that  may  have  been 
upon  its  surface. 

In  beginning  the  quantitative  analy- 
sis of  water  with  which  one  is  not  ac- 
quainted certain  preliminary  steps  are 
essential. 

It  is  necessary  to  know  approximately 
the  number  of  organisms  contained  in 
any  fixed  volume,  so  as  to  determine  the 
quantity  of  water  to  be  employed  for 
the  plates  or  tubes.  This  is  usually  done 
by  making  preliminary  plates  from  one 
drop,  two  drops,  0.25  c.c.,  0.5  c.c.,  and 
1  c.c.  of  the  water.  After  each  plate 
has  been  labelled  with  the  amount  of 
water  used  in  making  it,  it  is  placed 
aside  for  development.  When  this  has 
occurred  one  selects  the  plate  upon  which 
the  colonies  are  only  moderate  in  number 
— about  200  to  300  colonies  presenting 
— and  employs  in  the  subsequent  analysis  the  same  amount 
of  water  that  was  used  in  making  this  plate. 

If  the  original  water  contained  so  many  organisms  that 


Bottle  for  collecting 
water. 


652     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

there  developed  on  a  plate  or  tube  made  with  one  drop  too 
many  colonies  to  be  easily  counted,  then  the  sample  must 
be  diluted  with  one,  ten,  twenty-five,  fifty,  or  one  hundred 
volumes,  as  the  case  may  require,  of  sterilized  distilled  water. 
This  dilution  must  be  accurate,  and  its  exact  extent  noted, 
so  that  subsequently  the  number  of  organisms  per  volume 
in  the  original  water  may  be  calculated. 

The  use  of  a  drop  is  not  sufficiently  accurate.  The  dilu- 
tion should  therefore  always  be  to  a  degree  that  will  admit 
of  the  employment  of  a  volume  of  water  that  may  be  exactly 
measured,  0.25  and  0.5  c.c.  being  the  amounts  most  con- 
venient for  use. 

Duplicate  plates  should  always  be  made,  arid  the  mean 
of  the  number  of  colonies  that  develop  upon  them  taken 
as  the  basis  from  which  to  calculate  the  number  of  organ- 
isms per  volume  in  the  original  water. 

For  example:  from  a  sample  of  water  0.25  c.c.  is  added 
to  a  tube  of  liquefied  gelatin,  carefully  mixed  and  poured  as 
a  plate.  When  development  occurs  the  number  of  colonies 
is  too  numerous  to  be  accurately  counted.  One  cubic  cen- 
timeter of  the  original  water  is  then  to  have  added  to  it, 
under  precautions  that  prevent  contamination  from  with- 
out, 99  c.c.  of  sterilized  distilled  water — that  is,  we  have  now 
a  dilution  of  1  :  100.  Again,  0.25  c.c.  of  this  dilution  is 
plated,  and  we  find  180  colonies  on  the  plate.  Assuming 
that  each  colony  develops  from  an  individual  bacterium, 
though  this  is  perhaps  not  strictly  true,  we  had  180  organ- 
isms in  0.25  c.c.  of  our  1  :  100  dilution;  therefore  in  0.25 
c.c.  of  the  original  water  we  had  180X100  =  18,000  bacteria, 
which  will  be  72,000  bacteria  per  cubic  centimeter  (0.25  c.c. 
=  18,000,  1  c.c.  =  18,000x4  =  72,000).  The  results  are 
always  to  be  expressed  in  terms  of  the  number  of  bacteria 
per  cubic  centimeter  of  the  original  water. 


BACTERIOLOGICAL  STUDY  OF   WATER 


653 


Another  point  of  very  great  importance  (already  men- 
tioned) is  the  effect  of  temperature  upon  the  number  of 
colonies  of  bacteria  that  will  develop  on  the  plates  made 
from  water.  It  must  always  be  remembered  that  a  larger 
number  of  colonies  appear  on  gelatin  plates  made  from  water 
and  kept  at  18°  to  20°  C.  than  on  agar-agar  plates  kept  in 
the  incubator.  The  following  table,  illustrative  of  this 
point,  gives  the  results  of  parallel  analyses  of  the  same  waters, 
the  one  series  of  counts  having  been  made  upon  gelatin 
plates  at  the  ordinary  temperature  of  the  room,  the  other 
upon  plates  of  agar-agar  kept  for  the  same  length  of  time 
in  the  incubator  at  from  37°  to  38°  C.  It  will  be  seen  from 
the  table  that  much  the  larger  number  of  colonies — i.  e., 
much  higher  results — were  always  obtained  when  gelatin  was 
employed.  The  importance  of  this  point  in  the  quantita- 
tive bacteriological  analysis  of  water  is  too  apparent  to 
require  further  comment. 

TABLE  COMPARING  THE  RESULTS  OBTAINED  BY  THE  USE  OF  GELATIN  AT 
18°-20°  C.  AND  AGAR-AGAR  AT  37°-38°  C.  IN  QUANTITATIVE  BAC- 
TERIOLOGICAL ANALYSES  OF  WATER.  RESULTS  RECORDED  ARE  THE 
NUMBER  OF  COLONIES  THAT  DEVELOPED  FROM  THE  SAME  AMOUNT  OF 
VARIOUS  WATERS  IN  EACH  SERIES.1 

NUMBER  OF  COLONIES  FROM  WATER  THAT  DEVELOPED  UPON — 
Gelatin  plates  at  18°  to  20°  C.  Agar-agar  plates  at  37°  to  38°  C. 

310 170 

280      .  140 

310 180 

340  160 

650  210 

630  320 

380  290 

400  210 

1000  .      . 100 

890  130 

340  280 

370  210 

490  110 

580  / 100 

1  I  am  indebted  to  James  Homer  Wright,  Thomas  Scott  Fellow  in  Hygiene 
1892-1893),  University  of  Pennsylvania,  for  the  results  presented  in  this 
table. 


654     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

Another  point  of  equal  importance  in  its  influence  upon 
the  number  of  colonies  that  develop  is  the  reaction  of  the 
gelatin.  A  marked  excess  of  either  alkalinity  or  acidity 
always  has  a  retarding  effect  upon  many  species  found  in 
water.  Fuller's  experience  at  the  Lawrence  (Mass.)  Ex- 
periment Station  has  shown  that  gelatin  of  such  a  degree 
of  acidity  as  to  require  the  further  addition  of  from  15  to 
20  c.c.  per  liter  of  a  normal  caustic  alkali  solution  to  bring 
it  to  the  phenolphthalein  neutral  point  gives,  on  the  whole, 
the  best  results.  Thus,  by  way  of  illustration,  Fuller  found 
that  a  sample  of  Merrimac  River  water  gave  5800  colonies 
per  c.c.  on  phenolphthalein  neutral  gelatin,  15,000  colonies 
on  gelatin  that  would  need  20  c.c.  of  normal  alkali  solution 
to  bring  it  up  to  the  phenolphthalein  neutral  point — i.  e., 
a  feebly  acid  nutrient  gelatin,  and  500  colonies  on  a  gelatin 
so  alkaline  as  to  require  20  c.c.  of  a  normal  acid  solution 
to  bring  it  back  to  the  phenolphthalein  neutral  point. 

Throughout  this  part  of  the  work  it  is  to  be  borne  in 
mind  that  when  reference  is  made  to  plates  it  is  not  to  a 
set,  as  in  isolation  experiments,  but  to  a  single  plate. 

Method  of  Counting  the  Colonies  on  Plates. — For  conven- 
ience in  counting  colonies  on  plates  or  in  tubes  it  is  customary 
to  divide  the  whole  area  of  the  gelatin  occupied  by  colonies 
into  smaller  areas,  and  either  count  all  the  colonies  in  each 
of  these  areas  and  add  the  several  sums  together  for  the 
total,  or  to  count  the  number  of  colonies  in  each  of  several 
areas,  ten  or  twelve,  take  the  mean  of  the  results  and  multiply 
this  by  the  number  of  areas  containing  colonies.  The  latter 
procedure  obtains,  of  course,  only  when  all  the  areas  are 
of  the  same  size.  By  this  method,  however,  the  results 
vary  so  much  in  different  counts  of  the  same  plate  that  they 
cannot  be  considered  as  more  than  rough  approximations. 


BACTERIOLOGICAL  STUDY  OF   WATER  655 

NOTE. — Prepare  a  plate;  calculate  the  number  of  colonies 
upon  it  by  this  latter  method.  Now  repeat  the  calcula- 
tion, making  the  average  from  another  set  of  squares.  Now 
actually  count  the  entire  number  of  colonies  on  the  plate. 
Compare  the  results. 

For  facilitating  the  counting  of  colonies  several  very 
convenient  devices  exist. 

Wolffhugel's  Counting-apparatus.— This  apparatus  (Fig. 
110)  consists  of  a  flat  wooden  stand,  the  centre  of  which  is 

FIG.  110 


Wolffhiigel's  apparatus  for  counting  colonies. 

cut  out  in  such  a  way  that  either  a  black  or  white  glass 
plate  may  be  placed  in  it.  These  form  a  background  upon 
which  the  colonies  may  more  easily  be  seen  when  the  plate 
to  be  counted  is  placed  upon  it.  When  the  gelatin  plate 
containing  the  colonies  has  been  placed  upon  this  back- 
ground of  glass  it  is  covered  by  a  transparent  glass  plate 
which  swings  on  a  hinge.  This  plate,  which  is  ruled  in 
square  centimeters  and  subdivisions,  when  in  position  is 
just  above  the  colonies,  without  touching  them.  The  gelatin 


656     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

plate  is  moved  about  until  it  rests  under  the  center  of  the 
area  occupied  by  the  ruled  lines.  The  number  of  colonies 
in  each  square  centimeter  is  then  counted,  and  the  sum 
total  of  the  colonies  in  all  these  areas  gives  the  number  of 
colonies  on  the  plate;  or,  as  has  already  been  indicated,  if 
the  number  of  colonies  be  very  great,  a  mean  may  be  taken 
of  the  number  in  several  (six  or  eight)  squares;  this  is  to 
be  multiplied  by  the  total  number  of  squares  occupied  by 
the  gelatin.  The  result  is  an  approximation  of  the  total 
number  of  colonies. 

When  the  colonies  are  quite  small,  as  is  frequently  the 
case,  the  counting  may  be  rendered  easier  by  the  use  of  a 
small  hand  lens.  (Fig.  111.) 

FIG.  Ill 


Lens  for  counting  colonies. 

Several  useful  modifications  of  the  apparatus  of  Wolff- 
hiigel  have  been  introduced.  The  most  important  is  that 
of  Lafar.1  Lafar's  counter  consists  of  a  glass  disk  of  the 
diameter  of  ordinary  size  Petri  dishes.  It  is  supplied  with 
a  collar  or  flange  that  fits  around  the  bottom  of  the  Petri 
dish,  and  thus  holds  the  counter  in  position.  The  disk  is 
ruled  with  concentric  circles,  and  its  area  is  divided  into 
sectors  of  such  sizes  that  the  spaces  between  the  concentric 
circles  and  the  radii  forming  the  sectors  are  of  equal  size. 

1  Centralblatt  fur  Bakteriologie  und  Parasitenkunde,  1891,  Bd.  xv,  S.  331 . 


BACTERIOLOGICAL  STUDY  OF  WATER  657 

Three  of  the  sectors  are  subdivided  into  smaller  areas  of 
equal  size  for  convenience  in  counting  when  the  colonies 
are  very  numerous.  The  principles  involved  are  similar 
to  those  of  the  preceding  apparatus,  but  the  circular  form 
of  the  apparatus  admits  of  more  exactness  when .  counting 
colonies  on  a  circular  plate.1 


8 
Fakes'  apparatus  for  counting  colonies  (reduced  one-third). 

Pakes2  has  introduced  a  cheap  and  convenient  modifi- 
cation of  Lafar's  apparatus.  It  consists  of  a  sheet  of  white 
paper  on  which  is  printed  a  black  disk  ruled  with  white 
lines,  in  somewhat  the  same  fashion  as  is  Lafar's  counter, 

1  Lafar's  apparatus  is  to   be  obtained  from   F.   Mollenkopf,   10  Thor- 
strasse,  Stuttgart,  who  holds  the  patent  for  it.    Its  price  is  about  8  marka. 

2  Journal  of  Bacteriology  and  Pathology,  1896,  iv,  No.  1. 

42 


658     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

though  the  areas  of  the  smallest  subdivisions  are  not  of  one 
size  and  do  not  bear  a  constant  relation  to  each  other.1  To 
use  this  apparatus  (Fig.  112)  the  Petri  dish  is  placed  cen- 
trally upon  it,  the  cover  of  the  dish  is  removed,  and  the 
colonies  are  counted  as  they  lie  over  the  spaces  bounded  by 
the  white  lines  on  the  black  disk  beneath.  When  the  plate 
is  centered  over  the  black  disk  the  portion  lying  over  one 
sector  is  exactly  one-sixteenth  of  the  whole  plate. 

FIG.  113 


Esmarch's  apparatus  for  counting  colonies  in  rolled  tubes. 

Esmarch's  Counter. — Esmarch  devised  a  counter  (Fig. 
113)  for  estimating  the  number  of  colonies  present  upon 
a  cylindrical  surface,  as  when  in  rolled  tubes.  The  prin- 
ciples and  methods  of  estimation  are  practically  the  same 
as  those  given  for  Wolffhugel's  apparatus. 

1  Copies  of  this  apparatus  are  to  be  had  of  Ash  &  Co.,  42  Southwark 
Street,  London,  or  of  Lentz  &  Sons,  North  Eleventh  Street,  Philadelphia, 
Pa.  (The  cost  is  but  a  few  cents  per  copy.) 


BACTERIOLOGICAL  STUDY  OF   WATER   '        659 

A  simpler  method  than  by  the  use  of  Esmarch's  apparatus 
may  be  employed  for  counting  the  colonies  in  rolled  tubes. 
It  consists  in  dividing  the  tube  by  lines  into  four  or  six 
longitudinal  areas,  which  are  subdivided  by  transverse 
lines  about  1  or  2  cm.  apart.  The  lines  may  be  drawn  with 
pen  and  ink.  They  need  not  be  exactly  the  same  distance 
apart  nor  exactly  straight.  Beginning  with  one  of  these 
squares  at  one  end  of  the  tube,  which  may  be  marked  with 
a  cross,  the  tube  is  twisted  with  the  fingers,  always  in  one 
direction,  and  the  exact  number  of  colonies  in  each  square 
as  it  appears  in  rotation  is  counted,  care  being  taken  not 
to  count  a  square  more  than  once;  the  sums  are  then  added 
together,  and  the  result  gives  the  number  of  colonies  in 
the  tube.  This  method  may  be  facilitated  by  the  use  of  a 
hand-lens. 

In  all  these  methods  there  is  one  error  difficult  to  eliminate : 
it  is  assumed  that  each  colony  has  grown  from  a  single 
organism.  This  is  probably  not  always  the  case,  as  there 
may  exist  clumps  of  bacteria  which  represent  hundreds  or 
even  thousands  of  individuals,  but  which  still  give  rise  to 
but  a  single  colony — obviously  this  is  of  necessity  estimated 
as  a  single  organism  in  the  water  under  analysis. 

Where  grounds  exist  for  suspecting  the  presence  of  these 
clumps  they  may  in  part  be  broken  up  by  shaking  the 
original  water  with  sterilized  sand. 

What  has  been  said  for  the  bacteriological  examination 
of  water  holds  good  for  all  fluids  which  are  to  be  subjected 
to  this  form  of  analysis. 

The  Sewage  Streptococcus. — Houston1  reached  the  con- 
clusion that  there  is  constantly  present  in  sewage  a  particular 
form  of  streptococcus  which  is  really  more  positively  indica- 

5Ann.  Report,  Local  Gov.  Board,  xxviii. 


660     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

live  of  the  contamination  of  water  by  sewage  than  is  bacillus 
coli.  This  opinion  was  under  investigation  by  members 
of  the  staff  of  the  Massachusetts  Institute  of  Technology, 
who  reached  the  conclusion  that  considerable  reliance  can 
be  placed  upon  the  presence  of  this  organism  as  an  indication 
of  sewage  pollution  of  water. 

The  presence  of  the  sewage  streptococcus  is  most  readily 
shown  in  the  sediment  in  fermentation  tubes  inoculated 
with  water  under  examination.  If  the  sewage  streptococcus 
is  present  it  is  very  easy  to  demonstrate  it  by  microscopic 
examination  of  the  sediment  after  twenty-four  to  forty- 
eight  hours.  In  addition  to  this  test  it  has  also  been  demon- 
strated by  Winslow1  that  the  estimation  of  the  degree  of 
acidity  of  the,  contents  of  the  fermentation  tube  is  a  safe 
indication  of  the  presence  of  the  sewage  streptococcus. 
When  this  organism  is  present  the  acidity  rises  far  more 
rapidly  and  to  a  greater  height  than  is  the  case  when  it  is 
absent,  so  that  in  this  way  an  additional  indicator  is  avail- 
able as  to  the  potability  of  a  water  under  examination. 

BACTERIOLOGICAL    ANALYSIS   OF   AIR. 

Quite  a  number  of  methods  for  the  bacteriological  study 
of  the  air  exist.  In  the  main  they  consist  either  in  allowing 
air  to  pass  over  solid  nutrient  media  (Koch,  Hesse)  and 
observing  the  colonies  which  develop  upon  the  media,  or 
in  filtering  the  bacteria  from  the  air  by  means  of  porous  and 
liquid  substances,  and  studying  the  organisms  thus  obtained. 
(Miguel,  Petri,  Strauss,  Wiirz,  Sedgwick-Tucker.)  Because 
of  their  greater  exactness,  the  latter  have  supplanted  the 
former  methods. 

1  Jour.  Med.  Research,  1902,  vol.  iii. 


BACTERIOLOGICAL  ANALYSIS  OF  AIR  661 

In  some  of  the  methods  which  provide  for  the  filtration 
of  bacteria  from  the  air  by  means  of  liquid  substances  a 
measured  volume  of  air  is  aspirated  through  liquefied 
gelatin;  this  is  then  rolled  into  an  Esmarch  tube  and  the 
number  of  colonies  counted,  just  as  is  done  in  water  analysis. 
This  is  the  simplest  procedure.  An  objection  sometimes 
raised  against  it  is  that  organisms  may  be  lost,  and  not 
come  into  the  calculation,  by  passing  through  the  medium 

FIG.  114 


Petri's  apparatus  for  bacteriological  analysis  of  air.    The  tube  packed  with 
sand  is  seen  at  the  point  a. 


in  the  center  of  an  air-bubble  without  being  arrested  by 
the  fluid — an  objection  that  appears  to  have  more  of  specu- 
lative than  of  real  value.  Filtration  through  porous  sub- 
stances appears,  on  the  whole,  to  give  the  best  results. 
Petri  recommends  aspiration  of  a  measured  volume  of  air 
through  glass  tubes  into  which  sterilized  sand  is  packed. 
(Fig.  114.)  When  aspiration  is  finished  the  sand  is  mixed 
with  liquefied  gelatin,  plates  are  made,  and  the  number  of 


662     APPLICATION  OF  METHODS  OF   BACTERIOLOGY 

developing  colonies  counted,  the  results  giving  the  number 
of  organisms  contained  in  the  volume  of  air  aspirated  through 
the  sand. 

The  main  objection  to  this  method  is  the  possibility  of 
mistaking  a  sand-granule  for  a  colony.  This  objection  has 
been  overcome  by  Sedgwick  and  Tucker,  who  employ 
granulated  sugar  instead  of  sand;  this,  when  brought  into 
the  liquefied  gelatin,  dissolves,  and  no  such  error  as  that 
possible  in  the  Petri  method  can  be  made. 

Sedgwick-Tucker  Method. — On  the  whole,  the  method 
proposed  by  Sedgwick  and  Tucker  gives  such  uniform 
results  that  it  is  to  be  preferred  to  others.  It  is  as  follows: 

The  apparatus  employed  by  them  consists  essentially  of 
three  parts: 

1.  A  glass  tube  of  special  form,  to  which  the  name  aerobio- 
scope  has  been  given. 

2.  A  stout  copper  cylinder  of  about  sixteen  litres  capacity, 
provided  with  a  vacuum-gauge. 

3.  An  air-pump. 

The  aerobioscope  (Fig.  115)  is  about  35  cm.  in  its  entire 
length;  it  is  15  cm.  long  and  4.5  cm.  in  diameter  at  its 
expanded  part;  one  end  of  the  expanded  part  is  narrowed 
to  a  neck  2.5  cm.  in  diameter  and  2.5  cm.  long.  To  the  other 
end  is  fused  a  glass  tube  15  cm.  long  and  0.5  cm.  inside 
diameter,  in  which  is  to  be  placed  the  filtering-material. 

Upon  this  narrow  tube,  5  cm.  from  the  lower  end,  a 
mark  is  made  with  a  file,  and  up  to  this  mark  a  small  roll 
of  brass-wire  gauze  (a)  is  inserted;  this  serves  as  a  stop 
for  the  filtering-material  which  is  to  be  placed  over  it. 
Beneath  the  gauze  (at  6),  and  also  at  the  large  end  (c), 
the  apparatus  is  plugged  with  cotton.  When  thoroughly 
cleaned,  dried,  and  plugged,  the  apparatus  is  to  be  steril- 


•     BACTERIOLOGICAL  ANALYSIS  OF  AIR  663 

ized  in  the  hot-air  sterilizer.  When  cool  the  cotton  plug  is 
removed  from  the  large  end  (c),  and  thoroughly  dried  and 
sterilized  No.  50  granulated  sugar  is  poured  in  until  it  just 
fills  the  10  cm.  (d)  of  the  narrow  tube  above  the  wire  gauze. 
This  column  of  sugar  is  the  filtering-material  employed  to 
engage  and  retain  the  bacteria.  After  pouring  in  the  sugar 
the  cotton-wool  plug  is  replaced,  and  the  tube  is  again 
sterilized  at  120°  C.  for  several  hours. 

Taking  the  air  sample.  In  order  to  measure  the  amount 
of  air  used  the  value  of  each  degree  on  the  vacuum-gauge 
is  determined  in  terms  of  air  by  means  of  an  air-meter,  or 
by  calculation  from  the  known  capacity  of  the  cylinder. 

FIG.  115 


c  e  da 

The  Sedgwick-Tucker  aerobioscope. 

This  fact  ascertained,  the  negative  pressure  indicated  by 
the  needle  on  exhausting  the  cylinder  shows  the  volume  of 
air  which  must  pass  into  it  in  order  to  fill  the  vacuum.  By 
means  of  the  air-pump  one  exhausts  the  cylinder  until  the 
needle  reaches  the  mark  corresponding  to  the  amount  of 
air  required.1 

A  sterilized  aerobioscope  is  now  to  be  fixed  in  the  upright 
position  and  its  small  end  connected  by  a*  rubber  tube 

1  Such  a  cylinder  and  air-pump  are  not  necessary.  A  pair  of  ordinary 
aspirating  bottles  of  known  capacity  graduated  into  liters  and  fractions 
thereof  answer  perfectly  well.  Or  one  can  determine  by  the  weight  of 
water  that  has  flowed  from  the  aspirator  the  volume  of  air  that  has  passed 
in  to  take  its  place — i.  e.,  the  volume  of  air  that  has  passed  through  the 
aerobioscppe. 


664     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

with  a  stopcock  on  the  cylinder,  or  to  a  glass  tube  tightly 
fixed  in  the  neck  of  an  aspirat ing-bottle  by  means  of  a 
perforated  rubber  stopper.  The  cotton  plug  is  then  moved 
from  the  upper  end  of  the  aerobioscope,  and  the  desired 
amount  of  air  is  aspirated  through  the  sugar.  Dust-par- 
ticles and  bacteria  will  be  held  back  by  the  sugar.  During 
manipulation  the  cotton  plug  is  to  be  protected  from  con- 
tamination. 

FIG.  116 


Bent  funnel  for  use  with  aerobioscope. 

When  the  required  amount  of  air  has  been  aspirated 
through  the  sugar  the  cotton  plug  is  replaced,  and  by  gently 
tapping  the  aerobioscope  while  held  in  an  almost  horizontal 
position  the  sugar,  and  with  it,  the  bacteria,  are  brought 
into  the  large  part  (e)  of  the  apparatus..  When  all  the  sugar 
is  thus  shaken  down  into  this  part  of  the  apparatus  about 
20  c.c.  of  liquefied,  sterilized  gelatin  is  poured  in  through 
the  opening  at  the  end  c,  the  sugar  dissolves,  and  the  whole 


BACTERIOLOGICAL  STUDY  OF  THE  SOIL          G65 

is  then  rolled  on  ice,  just  as  is  done  in  the  preparation  of 
an  ordinary  Esmarch  tube. 

The  gelatin  is  most  easily  poured  into  the  aerobioscope 
by  the  use  of  a  small,  sterilized,  cylindrical  funnel  (Fig. 
116),  the  stem  of  which  is  bent  to  an  angle  of  about  110 
degrees  with  the  long  axis  of  the  body. 

The  larger  part  of  the  aerobioscope  is  divided  into  squares 
to  facilitate  the  counting  of  the  colonies. 

By  the  employment  of  this  apparatus  one  can  filter  the 
air  at  any  place,  and  can  then,  without  fear  of  contamination, 
carry  the  tubes  to  the  laboratory  and  complete  the  analysis. 
Aside  from  this  advantage,  the  filter  being  soluble  only  the 
insoluble  bacteria  are  left  imbedded  in  the  gelatin. 

For  general  use  this  method  is  to  be  preferred  to  the 
others  that  have  been  mentioned. 


BACTERIOLOGICAL   STUDY   OF   THE   SOIL. 

Bacteriological  study  of  the  soil  may  be  made  by  either 
breaking  up  small  particles  of  earth  in  liquefied  media  and 
making  plates  directly  from  this;  or  by  what  is  perhaps 
a  better  method,  as  it  gets  rid  of  insoluble  particles  which 
may  give  rise  to  errors;  breaking  up  the  soil  in  sterilized 
water  and  then  making  plates  immediately  from  the  water. 

It  must  be  borne  in  mind  that  many  of  the  ground-organ- 
isms belong  to  the  anaerobic  group,  so  that  in  these  studies 
this  point  should  be  remembered  and  the  methods  for  the 
cultivation  of  such  organisms  practised  in  connection  with 
the  ordinary  methods.  It  must  also  be  remembered  that 
the  nitrifying  organisms,  everywhere  present  in  the  ground, 
cannot  be  isolated  by  the  ordinary  methods,  and  will  not 


666     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

appear  in  plates  made  after  either  of  the  above  plans.  The 
special  devices  for  their  cultivation  are  described  in  the 
chapter  on  Soil-organisms. 

BACTERIOLOGICAL   STUDY   OF   MILK. 

The  possibility  of  milk  serving  as  a  vehicle  in  which 
disease-producing  bacteria  may  be  disseminated  through- 
out a  community  has  long  been  recognized,  and  epidemics 
of  typhoid  fever  have  been  traced  directly  to  infected  milk, 
while  such  diseases  as  diphtheria  and  scarlet  fever  are  also 
frequently  regarded  as  being  conveyed  in  the  same  manner. 

In  recent  years  the  detailed  study  of  the  milk  of  individ- 
ual cows  has  revealed  the  fact  that  streptococcus  mastitis 
is  not  an  uncommon  occurrence  in  herds,  and  it  has  fre- 
quently been  observed  that  milk  rich  in  streptococci  may 
prove  dangerous  when  fed  to  infants  and  convalescents. 

Since  milk  is  such  a  favorable  medium  for  the  growth  of 
a  variety  of  bacteria  it  is  not  at  all  uncommon  to  find  market 
milk  very  rich  in  bacteria,  especially  if  it  has  been  collected 
in  a  careless  manner  in  dirty  receptacles,  in  unsanitary 
stables,  and  has  been  shipped  long  distances  at  comparatively 
high  temperatures. 

For  these  various  reasons  the  bacteriological  study  of 
milk  has  gained  considerable  prominence  during  the  past 
few  years — ;so  much  so  that  in  some  localities  an  effort  is 
being  made  to  establish  a  bacterial  standard  for  market 
milk — that  is,  milk  containing  more  than  a  certain  number 
of  bacteria  is  not  regarded  as  suitable  for  use.  Whether 
such  a  standard  can  be  maintained  or  not  remains  to  be 
demonstrated.  The  several  milk  commissions  composed 
of  pediatrists  in  various  large  cities  have  established  a 


BACTERIOLOGICAL  STUDY  OF  MILK  667 

bacterial  standard  for  approved  milk  of  10,000  bacteria  to 
the  cubic  centimeter.  Experience  has  shown  that  it  is 
possible  to  market  milk  that  meets  this  bacterial  standard 
sometimes  with  merely  ordinary  precautions  with  regard 
to  cleanliness.  In  larger  dairies  it  has  frequently  been  a 
question  of  some  difficulty  on  account  of  the  elaborate 
scale  on  which  the  business  is  conducted. 

Quantitative  Bacteriological  Analysis. — In  the  quantitative 
bacteriological  examination  of  market  milk  it  is  necessary 
to  dilute  the  milk  with  sterile  water  or  sterile  salt  solution 
before  plating  on  account  of  the  very  large  numbers  of 
bacteria  present.  The  degree  of  dilution  that  is  necessary 
will  depend  upon  the  nature  of  the  dairy  from  which  the 
milk  is  derived,  the  age  of  the  milk,  and  the  temperature 
at  which  it  has  been  kept.  Usually  a  dilution  of  1  to  100, 
1  to  1000,  and  1  to  10,000  is  sufficient.  From  these  dilutions 
plate  cultures  are  made  with  0.1,  0.2,  0.3  cubic  centimeter 
of  each  dilution. 

Qualitative  Bacteriological  Analysis. — Aside  from  the 
quantitative  bacteriological  analysis  of  milk  the  qualita- 
tive analysis  has  received  a  great  deal  of  attention.  Detailed 
qualitative  analysis  necessarily  entails  an  enormous  amount 
of  labor,  but  the  detection  of  certain  forms  of  bacteria  is 
not  always  very  difficult.  This  applies  especially  to  the 
detection  of  streptococci. 

Since  milk  containing  streptococci  in  considerable  num- 
bers is  derived  from  the  udder  of  a  cow  suffering  from  some 
form  of  mastitis,  it  is  always  possible  to  find  pus  in  such 
milk.  Consequently  it  is  customary  to  examine  such  milk 
for  the  presence  of  both  streptococci  and  pus.  This  is  done 
by  centrifuging  a  cubic  centimeter  of  the  milk  and  collecting 
the  sediment  on  a  clean  cover-slip  and  staining  with  Loffler's 


668     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

methylene-blue.  In  this  manner  practically  all  the  sediment 
derived  from  one  cubic  centimeter  can  be  obtained  on  the 
cover-slip  and  a  fairly  satisfactory  estimate  can  be  made  of 
the  relative  number  of  pus  cells  in  this  quantity  of  milk  as 
well  as  at  the  same  time  an  estimation  of  the  relative  number 
of  streptococci. 

Milk  that  shows  pus  cells  along  with  distinct  chains  of 
streptococci,  either  extra-  or  intracellular,  is  usually  regarded 
as  dangerous  in  character,  and  boards  of  health  usually 
direct  that  the  cows  from  which  such  milk  is  derived  be 
excluded  from  the  dairy  until  such  time  as  the  milk  is  free 
from  these  elements. 


APPENDIX. 


LIST  of  apparatus  and  materials  required  in  a  beginner's 
bacteriological  laboratory : 

MICROSCOPE  AND  ACCESSORIES. 

Microscope  with  coarse  and  fine  adjustment  and  heavy, 
firm  base;  Abbe  sub-stage  condensing  system,  arranged 
either  as  the  "simple"  or  as  the  regular  Abbe  condenser, 
in  either  case  to  be  provided  with  iris  diaphragm;  objec- 
tives equivalent,  in  the  English  nomenclature,  to  about 
one-fourth  inch  and  one-sixth  inch  dry,  and  one-twelfth 
inch  oil-immersion  system;  a  triple  revolving  nose-piece; 
three  oculars,  varying  in  magnifying  power;  and  a  bottle 
of  immersion  oil. 

Glass  slides,  English  shape  and  size  and  of  colorless  glass. 

Six  slides  with  depressions  of  about  1  cm.  in  diameter  in 
centre. 

Cover-slips,  15  by  15  mm.  square  and  not  more  than  from 
0.15  to  O.L8  mm.  thick. 

Forceps.  One  pair  of  fine-pointed  forceps  and  one  pair 
of  the  Cornet  or  Stewart  pattern,  for  holding  coverslips. 

Platinum  needles  with  glass  handles.  One  straight, 
about  4  cm.  long;  one  looped  at  the  end,  about  4  cm.  long; 
and  one  straight,  about  8  cm.  long.  Glass  handles  to  be 
about  3  mm.  in  thickness  and  from  15  to  17  cm.  long. 

(669) 


670     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 
STAINING-  AND  MOUNTING-REAGENTS. 

200  c.c.  of  saturated  alcoholic  solution  of  fuchsin. 

200  c.c.  of  saturated  alcoholic  solution  of  gentian-violet. 

200  c.c.  of  saturated  alcoholic  solution  of  methylene-blue. 

200  grams  of  pure  aniline. 

200  grams  of  C.  P.  carbolic  acid. 

500  grams  of  C.  P.  nitric  acid. 

500  grams  of  C.  P.  sulphuric  acid. 

200  grams  of  C.  P.  glacial  acetic  acid. 

1  liter  of  ordinary  93-95  per  cent,  alcohol. 

1  liter  of  absolute  alcohol. 
500  grams  of  ether. 

500  grams  of  pure  xylol. 
50  grams  of  Canada  balsam  dissolved  in  xylol. 
100  grams  of  Schering's  celloidin. 

10  grams  of  iodine  and  30  grams  of  potassium  iodide  in 
substance. 

100  grams  of  tannic  acid. 
100  grams  of  ferrous  sulphate. 
Distilled  water. 

FOR  NUTRIENT  MEDIA. 

J  pound  of  beef-extract. 
250  grams  of  peptone. 

2  kilograms  of  first  quality  gelatin. 
100  grams  of  agar-agar  in  substance. 

200  grams  of  sodium  chloride  (ordinary  table-salt). 
500  grams  of  pure  glycerin. 
50  grams  of  pure  glucose. 
20  grams  of  pure  lactose. 
100  grams  of  caustic  potash. 


APPENDIX  671 

200  c.c.  of  litmus  tincture. 

10  grams  of  rosolic  acid  (corallin). 

Blue  and  red  litmus-paper;  curcuma  paper. 

5  grams  of  phenolphthalein  in  substance. 
Filter-paper,  the  quality  ordinarily  used  by  druggists. 
100  grams  of  pyrogallic  acid. 

1  kilogram  of  C.  P.  granulated  zinc. 

GLASSWARE. 

200  best  quality  test-tubes,  slightly  heavier  than  those 
used  for  chemical  work,  about  12  to  13  cm.  long  and  12  to 
14  mm.  inside  diameter. 

15  Petri  double  dishes  about  8  or  9  cm.  in  diameter  and 
from  1  to  1.5  cm.  deep. 

6  Florence  flasks,  1000  c.c.  capacity. 
6  Florence  flasks,  500  c.c.  capacity. 

12  Erlenmeyer  flasks,  100  c.c.  capacity. 

1  graduated  measuring-cylinder,  1000  c.c.  capacity. 

1  graduated  measuring-cylinder,  100  c.c.  capacity. 

25  bottles,  125  c.c.  capacity,  narrow  necks  with  ground- 
glass  stoppers. 

25  bottles,  125  c.c.  capacity,  wide  mouths,  with  ground- 
glass  stoppers. 

1  anatomical  or  preserving  jar,  with  tightly  fitting  cover, 
of  about  4  liters  capacity,  for  collecting  blood-serum. 

2  battery  jars  of  about  2  liters  capacity,  provided  with 
loosely  fitting,  weighted,  wire-net  covers  for  mice. 

10  feet  of  soft-glass  tubing,  2  or  3  mm.  inside  diameter. 
20  feet  of  soft-glass  tubing,  4  mm.  inside  diameter. 
6  glass  rods,  18  to  20  cm.  long  and  3  or  4  mm.  in  diameter. 
6  pipettes  of  1  c.c.  each,  divided  into  tenths. 


672     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

2  pipettes  of  10  c.c.  each,  divided  into  cubic  centimeters 
and  fractions. 

1  burette  of  50  c.c.  capacity,  divided  into  cubic  centimeters 
and  fractions. 

1  separatirig-funnel  of  750  c.c.  capacity  for  filling  tubes. 

2  glass  funnels,  best  quality,  about  15  cm.  in  diameter. 
2  glass  funnels,  best  quality,  about  8  cm.  in  diameter. 

2  glass  funnels,  best  quality,  about  4  or  5  cm.  in  diameter. 

2  porcelain  dishes,  200  c.c.  capacity. 

6  ordinary  water  tumblers  for  holding  test-tubes. 

1  ruled  plate  for  counting  colonies. 

1  gas-generator,  600  c.c.  capacity,  pattern  of  Kipp  or  v. 
Wartha. 

BURNERS,   TUBING,   ETC. 

2  Bunsen  burners,  single  flame. 
1  Rose-burner. 

1  Koch  safety-burner,  single  flame. 
6  feet  of  white-rubber  gas-tubing. 

12  feet  of  pure  red-rubber  tubing,  5  to  6  mm.  inside 
diameter. 

1  thermo-regulator,  pattern  of  L.  Meyer  or  Reichert. 

2  thermometers,    graduated   in   degrees   of   Centigrade, 
registering  from  0°  to  100°  C.,  graduated  on  the  stem. 

1  thermometer  graduated  in  tenths  and  registering  from 
0°  to  50°  C. 

1  thermometer  registering  to  200°  C. 

INSTRUMENTS,   ETC. 

1  microtome,  pattern  of  Schanze,  with  knife. 
1  razor-strop. 


APPENDIX  673 

6  cheap-quality  scalpels,  assorted  sizes. 
2  pair  heavy  dissecting-forceps. 
1  pair  medium-size  straight  scissors. 
1  pair  small-size  straight  scissors. 

1  hypodermic  syringe  that  will  stand  steam  sterilization. 

2  teasing-needles. 

1  pair  long-handled  crucible-tongs  for  holding  mice. 

1  wire  mouse-holder. 

2  small  pine  boards  on  which  to  tack  animals  for  autopsy. 
2  covered  stone  jars  for  disinfectants  and  for  receiving 

infected  materials. 


INCUBATORS  AND   STERILIZERS. 

1  incubator,  simple  square  form,  either  entirely  of  copper 
or  of  galvanized  iron  with  copper  bottom. 

1  medium-size  hot-air  sterilizer  with  double  walls,  asbes- 
tos jacket,  and  movable  false  bottom  of  copper  plates. 

1  medium-size  steam  sterilizer;  either  the  pattern  of 
Koch  or  that  known  as  the  Arnold  steam  sterilizer,  prefer- 
ably the  latter. 

MISCELLANEOUS. 

1  pair  of  balances,  capacity  1  kilogram;  accurate  to  0.2 
grams. 

1  set  of  cork-borers. 
1  hand-lens. 

1  wooden  filter-stand. 

2  iron  stands  with  rings  and  clamps. 

3  round,  galvanized  iron-wire  baskets  to  fit  loosely  into 
steam  sterilizer. 

43 


674     APPLICATION  OF  METHODS  OF  BACTERIOLOGY 

3  square,  galvanized  iron-wire  baskets  to  fit  loosely  into 
hot-air  sterilizer. 

1  sheet-iron  box  for  sterilizing  pipettes,  etc. 

1  covered  agate-ware  saucepan,  1200  c.c.  capacity. 

2  iron  tripods. 

1  yard  of  moderately  heavy  wire  gauze. 

2  test-tube  racks,  each  holding  24  tubes,  12  in  a  row. 

1  constant-level,  cast-iron  water-bath. 

2  potato-knives. 

2  test-tube  brushes  with  reed  or  wire  handles. 

Cotton-batting. 

Copper  wire,  wire  nippers. 

Round  and  triangular  files. 

Labels. 

Towels  and  sponges. 


INDEX. 


A 


ABSCESSES,  350-352 
Acid  proof  bacteria,  449 

actinomycotic  growth,  453 
cultivation  of,  451 
distinguished  from  bacillus 

tuberculosis,  451 
distribution  of,  450 
inoculation  with,  452 
mycelial  growth,  453 
relation   to   bacillus   tuber- 
culosis, 454 
staining  of,  449 
Actinomycetes,  457 
bovis,  459 
Eppingeri,  467 
fascinicus,  465 
madurse,  463 
pathogenesis  of,    459,  463,  466, 

467,  468 

pseudotuberculosis,  468 
Aerobic  bacteria,  55,  227 

cultivation       of,      Buchner's 

method,  227 
Frankel's  method,  227 
Kitasato  and  Weil's  method, 

230 

Park's  method,  230 
Aerobioscope,  663 
Agar-agar,  lactose-litmus,  142 
nutrient,  129 
filtration  of,  131 
preparation  of,  129 
Agglutinins,  299 
Aggressins,  299 

Air,  bacteriological  analysis  of,  660 
apparatus  for,  661-663 
methods  of,  661-665 
Alexin,  289 


Ammonia,  test  for,  225 
Anaerobic  bacteria,  55,  227 

method     of     cultivation     of, 

227 

Buchner's,  227 
Frankel's,  227 
Kitasato's,  230 
Park's,  230 
Animalculse,  20 
Animals,  cultures  from,  259 
blood,  260 
precautions,  260 
holders  for,  234-238 
inoculation  of,  232-254 
into  eye,  247 
into  lymphatics,  244 
into  serous  cavities,  245 
intravascular,  239 
subcutaneous,  252 
observation  of,  248-254 
postmortem  examination  of,  255- 

259 

cultures  at,  257 
Anthrax,    bacillus    of,     583.     See 

Bacillus  anthracis. 
discovery  of,  21 
immune  serum,  594 
protective  inoculation  against, 

591 

Antibodies,  303 

Antiseptics,  determination  of  pro- 
perties of,  339 
experiments  with,  340 
testing  of,  339 
Antitoxins,  273 

Apparatus  for  beginner's  labora- 
tory, 669-674 
Arnold's  sterilizer,  89 
Autoclave,  91 
Avian  tuberculosis,  455 

(675) 


676 


INDEX 


B 


BACILLI,  66 

Bacillus  aerogenes  capsulatus,  627 
cultivation  of,  627-628 
morphology  of,  627 
pathogenesis  of,  628 
anthracis,  583-599 

cultivation  of,  586-588 

discovery  of,  583 

experiments  with,  595-599 

immune  serum  from,  594 

inoculation  experiments,  589 

morphology  of,  583-585 

Pasteur's  vaccine,  591 

pathogenesis  of,  589 

protective  inoculation  against, 
591 

spore  formation  in,  584-586 

staining  of,  588 
Chauvei,  623-627 

cultivation  of,  623-625 

differentiation  of,  626 

discovery  of,  622 

morphology  of,  622 

occurrence  of,  621 

pathogenesis  of,  625 

spores  of,  622 
coli,  530 

bacillus  typhosus  and,  534 

cultivation  of,  532-535 

discovery  of,  530 

distribution  of,  531 

inoculation  experiments,  535 

morphology  of,  532 

pathogenesis  of,  531 
diphtherise,  482-498 

bacteria  simulating,  482 

cultivation  of,  480,  486-489 

differential  tests  for,  497-500 
stainings,  500 

experiments  with,  502 

isolation  of,  480-481 

loss  of  virulence  of,  494 

morphology  of,  482-486 

pathogenesis  of,  489-493 

staining  of,  489 

toxin  of,  493 
dysenteriae,  541-548 

agglutination  of,  546 

cultivation  of,  542 

discovery  of,  541 


Bacillus        dysenterise,       immune 

serum,  546 

inoculation  experiments,  544 
protective   inoculations   with, 

547 
types  of,  541-546 

Flexner,  541 

Harris,  546 

Hiss-Russell,  545 

Shiga,  542 

Strong,  545 
edematis,  616-620 

cultivation  of,  616-618 
discovery  of,  616 
how  to  obtain,  616,  619 
inoculation  experiments,  616 
morphology  of,  616 
pathogenesis  of,  619 
spores  of,  616 
where  found,  616 
enteriditis  sporogenes,  628 
influenzas,  424 

cultivation  of,  426 
demonstration  of,  426 
discovery  of,  425 
distribution  of,  425 
location  of,  428 
morphology  of,  426 
pathogenesis  of,  429 
vitality  of,  427 
leprae,  448 

characteristics  of,  448 
location  of,  in  tissues,  448 
microchemical   characters   of, 

449 

mallei  (of  glanders),  472 
agglutination  of,  478 
cultivation  of,  473 
inoculation  experiments,  475 
morphology  of,  472 
pathogenesis  of,  475-476 
resistance  of,  473 
staining  of,  476 
of  symptomatic  anthrax,  623 
paratyphosus,  538-540 
characteristics  of,  538 
discovery  of,  538 
r61e  of,  in  disease,  538 
pestis,  392 

antiserum,  399 
cultivation  of,  393-394 
discovery  of,  372  . 


INDEX 


677 


Bacillus  pestis,  flea  as  carrier  of, 
395 

history  of,  391 

inoculation  against,  397 

morphology  of,  392 

pathogenesis  of,  394 

portals  of  entry  of,  395-396 

resistance  of,  394 

staining  of,  393 

vaccination  in,  397 

variations  in,  395 
pseudotuberculosis,  457,  468 
pyocyaneus,  383.      See  Pseudo- 

monas  semginosa. 
smegmatis,  449 
sporogenes,  628 

cultivation  of,  629 

distribution  of,  628 

pathogenesis  of,  629 
tetani,  606-615 

antitoxin,  614 

colonies  of,  609 

cultivation  of,  609 

discovery  of,  606 

inoculation  experiments,  611 

morphology  of,  608 

pathogenesis  of,  611 

poisons  of,  612 

relation  of,  to  germicides,  610 

resistance  of,  610 

spores  of,  608,  610 

to  obtain,  606-608 
tuberculosis,  441.     See  Tubercu- 
losis. 

avium,  455 

general  characters  of,  455-457 
typhosus,  508-530 

agglutination  of,  515 

cultivation  of,  509-511 

in  drinking  water,  519 
isolation  from,  520 
methods  of,  520 

Endo-media  of,  524 

enriching  media  of,  521 

flagella  of,  509 

indol  production,  511 

inoculation  experiments,  513. 

isolation  of,  513,  520 

Drigolski-Conradi    method, 

522 

Endo  method,  524 
precipitation  method,  526 


Bacillus  typhosus,  morphology  of, 
508 

prophylactic  vaccination,  526 

staining  of,  509,  512 

in  tissues,  512 
Welchi,  627 
xerosis,  499 

Bacteria,  acid  proof,  449 
aerobic,  55 
anaerobic,  55,  227 
autolysis  of,  54 
biochemic  characters  of,  184 
chemotaxis  of,  60 
chromogenic,  37 
classification  of,  33 
colonies  of,  193 
commensal,  401,  413,  416 
composition  of,  65 
cooperating,  58 
cultures  of,  193-196 
definition  of,  33 
denitrifying,  39, 602 
discovery  of,  17 
electricity  and,  60 
enzymes  of,  44 

coagulating,  49 

diastatic,  49 

inverting,  49 

properties  of,  44,  51 

proteolytic,  48 

sugar  splitting,  50 
facultative,  55,  231 
flagella  of,  74-75 
grouping  of,  66 
importance  of,  35 
involution  of,  68-69 
isolation  of,  Koch's  observations 
on,  104 

principles  involved,  105 

in  pure  culture,  104,  110 
life  processes  of,  35 
light  and,  59 
metabolism  of,  33 
metatrophic,  34 
moisture  and,  59 
morphology  of,  63-66 
motility  of,  74 
multiplication  of,  66-70 
nitrifying,  38,  600-606 
nitrogen  fixing,  39,  600-606 
nutrition  of,  52-55 
natural,  33 


678 


INDEX 


Bacteria,  parasitic,  40-44 

specific  functions  of,  40-44 
paratrophic,  34 
pathogenic  properties  of,  197 
peculiarities  of,  33 
photogenic,  37 
place  in  nature,  35 
pressure  and,  59 
products  of,  53-54 
prototrophic,  34 
reduction  by,  223 
relation  to  oxygen,  55 
results  of  disease,  29 
saprogenic,  37 
saprophytic,  34-40 

specific  functions  of,  36-40 
size  of,  63 
spores  of,  72-74 
structure  of,  63-65 
symbiosis,  58 
systematic  study  of,  191 
bio-chemistry,  193-196 
biology,  193 
enzymes,  197 
gas  from,  197 
light  and,  196 
morphology,  192 
pathogenesis,  197 
pigment  and,  196 
reaction,  197 
temperature  and,  56 
thermal  death-point  of,  94 
thermophilic,  57 
thiogenic,  38 

,  toxins  of,  properties  of,  44 
variations  and  varieties,  199 

of  species,  199 
water,  57 
zymogenic,  38 
Bacteriacese,  33 
Bacterial      diseases,      vaccination 

against,  281 
proteins,  44 
toxins,  272 
examination   of   cultures   for, 

226 

types,  203 

Bacteriological  analysis  of  air,  660 
of  milk,  666 
of  soil,  665 
of  water,  637 
Bacteriology,  application  of,  323 


Bacteriology,  beginnings  of,  17-32 
birth  of  modern,  30-32 
historic  sketch  of,  17-32 
preliminary  experiments,   323 
Bacterium  Welchii,  627-628 
cultivation  of,  627 
discovery  of,  628 
morphology  of,  627 
pathogenesis  of,  628 
Bail,  293 

Behring-Kitasato,  290 
Billroth,  29,  30 
Biology  of  bacteria,  193 
Birsch-Hirschfeld,  26 
Blood  cultures,  260 
reactions  of  coagulation,  288 
Foder,  288 
Grohmann,  288 
immunity  from,  313 
Rauschenback,  288 
toxicity  of  alexin,  312 
Traube  and  Gscheidlen,  288 
serum  apparatus,   Rivas',   135- 

137 

from  small  animals,  135 
Loffler's,  143 
preparation  of,  133 
preservation  of,  138 
serum-water  mixture,  143 
Body,  defenses  of,  277 
Bonnet,  24 
Bordet,  299 

and  Gengou,  318 
Bouillon,  124 
Braatz,  335 
Brownian  motion,  210 
Brushes  for  cleaning  test-tubes,  146 
Buchner,  289 
Bumm,  370 
Burdon-Sanderson,  30 
Burner,  Koch's  safety,  159 


CHEMOTAXIS,  60 
Chevreul,  23 

Cholera,  Asiatic,  bacteria  of,  549- 
573 

antagonisms  of,  566 

cultivation  of,  552 

in  dead  bodies,  566 


INDEX 


679 


Cholera,  Asiatic,  bacteria  of,  dis- 
covery of,  549 
in  food,  566 
general    considerations    of, 

563 

grouping  of,  551 
immunity  from,  562 

Pfeiffer's   work   on,    562, 

563 

influences  of  acids  on,  558 
of  gases  on,  567 
of  light  on,  565 
inoculation    experiments, 

559 

morphology  of,  550 
pathogenesis  of,  561 
poison  of,  558,  562 
resistance  of,  567 
in  soil,  565 
in  water,  564 
when  dried,  567 
diagnosis  of,  568 
by  cultures,  569 
by  microscope,  569 
Classen,  27 
Cohn,  24 
Colon  bacillus,  530.     See  Bacillus 

coli. 

Colonies,  macroscopic  characteris- 
tics of,  165 
study  of,  165-167 
Colony-formation,  166 
Commensal  relations,  401,  413,  416 
Complement,  302-311,  316,  319- 

321 
fixation  of,  316 

applications  of,  319-321 
origin  of,  303 
specificity  of,  303 
Cooling  chamber  for  plates,   151, 

152 

Comma  bacillus,  549 
Corrosive  sublimate,  334 
Cover-slip  preparations,  172 
examination  of,  208-211 
hanging  block,  213 

drop,  209 
impression,  176 
staining  of,  171,  632 
steps  in  making  and  staining, 

172-176 
Culture  media,  111-144 


Cultures,  filtration  of,  226 
from  blood,  260 
gelatin,  214 
hanging  block,  213 

drop,  209      ' 
potato,  194,  215 
pure,  167 
smear,  167 
stab,  167 
test-tube,  167 


DARK-FIELD  illumination,  207 

Davaine,  21 

Decolorizing  solutions,  180 

Pappenheim's,  183 
Defenses  of  body,  277 
antibodies,  279 
antidotes,  278 
blood  serum,  279 
ciliated  epithelium,  277 
gastric  juice,  277 
germicidal  juices,  278 
orificial  hairs,  277 
phagocytic  cells,  278 
skin,  277 

Denitrification,  39,  602 
Dentrifying  bacteria,  38,  602 
Diphtheria,  480 
antitoxin,  503 

production  of,  504 
standardization  of,  505 
Behring,  505 
Ehrlich,  506 
bacillus   of,    482.    See   Bacillus 

diphtherias, 
bacteriological    diagnosis    of, 

480,  482 

variations  in,  483 
varieties  of,  493,  495,  496-497 
Diplococcus,  67 

intracellularis  meningitidis,  377. 
See  Micrococcus  intracellularis. 
Dishes,  Petri,  152 
Disinfectants,     determination     of 

properties  of,  331 
experiments  with,  340 
mode  of  action  of,  96-109 
testing  of,  331 

methods  of  precaution,  334 


680 


INDEX 


Disinfectants,  testing  of,  methods 
of  precaution,  Braatz's 
work,  335 

Geppert's  work,  335 
Disinfection,  95-103 

practical,  101 

Dry  heat,  experiments,  330 
DuckwalTs  staining  method,    189 
Durham's  fermentation  tube,  220 

solution,  140 
Dysentery,  541 
antiserum,  547 
bacillus  of,     541.    See    Bacillus 

dysenteriae. 

protective   inoculation    against, 
547 


EBERTH,  27 
Ehrlich,  27,  296 

and  Morgenroth,  300 

side  chain  theory  of,  296 
Emboli  of  micrococci,  351 
Endotoxins,  46,  274 

action  of,  276 

distinction  of,  from  toxins,  276 

immunity  for,  277 
Enzymes,  44-50 

coagulating,  49 

diastatic,  49 

inverting,  49 

proteolytic,  48 

sugar  splitting,  50 
Erysipelas,  359 
Esmarch's  counting  apparatus,  658 

tubes,  153 

Booker's  method,  154 
Experiments,  contact,  325 

exposure,  325 

sterilization,  327 


FACULTATIVE  bacteria,  55 
Fehleisen,  27 
Fermentation,  216 

early  views  on,  28 

tube,  218-221 
Durham's,  219 


erments,  44-45 

^ilter,  folding  of,  126 

Filterable  virus,  261 
nature  of,  265 
study  of,  263-265 
where  found,  265 

Filters,  bacterial,  226 

Fish,  296 

Fixation  of  complement,  316 

Flagella,  74-75 

staining  of,  187-189 

Flasks,  preparation  of,  145 

Flexner,  382 


GAS-PRESSURE  regulators,  163 
Gelatin,  agar  mixture,  144 

cultures,  214 

filtration  of,  126 

lactose-litmus,  142 

nutrient,  175 

preparation  of;  125 
Generation,  spontaneous,  22-25 
Geppert,  335 
Glanders,  470 

bacillus  of,    472.    See    Bacillus 
mallei. 

diagnosis  of,  478 
by  agglutination,  478 
by  mallein,  478 

pathology  of,  476 
Glassware,  preparation  of,  145 
Gonococcus,  370.  See  Micrococcus 

gonorrhoeas. 

Gonorrhea,  cause  of,  370 
Gram's  method  of  staining,  184 
Guarniari's  medium,  144 


HANGING-BLOCK  cultures,  213 

Hanging-drop  cultures,  209 

Haptophores,  301 

Harvey,  25 

Havins,  364-367 

Hemolysis,  312 

alien  blood  and,  312,  313 
Landois  on,  312 
mechanism  of,  314-316 


INDEX 


681 


Hemolytic  reaction,  312 

system,  315 
Henle,  21 
Hiss'  medium,  143 
Hoffman,  23 

Holders  for  animals,  234-238 
Hydrogen-ion  concentration,  115 
Hydrogen  sulphide,  test  for,  224 
Hypodermic  needle,  241 

syringes,  244 


ILLUMINATION,  dark-field,  207 
Immune  bodies,  303 
Immunity,  277-311 

acquired,  280 

active,  281 

alien  proteins,  299 

Behring  and  Kitasato  on,  290 

Buchner  on,  289 

Chauveau  on,  286 

conclusions  on,  305-311 

diverse  reactions  in,  302 

Ehrlich's  side-chain  theory,  300 
receptors,  300 

"exhaustion"  hypothesis,  287 

historic  sketch  of,  285 

Metchnikoff's  doctrine,  287 

natural,  280 

Nuttall's  work  in,  288 

opsonic  doctrine,  294 

passive,  281 

Pasteur's  doctrine,  287 

Pfeiffer's  reaction,  292 

phagocytosis,  287 

"retention"  hypothesis,  287 

Roux  and  Yersin  on,  290 

views  on,  304-311 

Wright  and  Douglass  on,  278 
Immunology,  beginnings,  28 
Incubator,  157 
Indicators,  119 
Indol  production,  221 

tests  for,  222 
Infection,  266 

mechanism  of,  272,  309 

types  of,  266-272 

views  on,  309-311 
Influenza,    bacillus    of,    424.    See 

Bacillus  influenzse. 


Influenza,  filterable  virus  in,  425 
historic  outbreaks  of,  424 
pneumococci  in,  425 
streptococci  in,  425 
Inoculation  methods,  232-254 
eye,  247 
lymphatics,  244 
needles  and  syringes  for,  241, 

244 

serous  cavities,  245 
subcutaneous,  232 
vessels,  239 

Intracellular  toxins,  46,  274 
Involution,  68 

Isolation  of  bacteria  in  pure  cul- 
ture, 104-110 

of  pathogenic  organisms  in  the 
sputum,  401 


KLEBS,  26,  27,  30 

Koch,  30 

Koch-Ehrlich  aniline  water  solu- 
tion, 178 

Koch's  postulates,  440 
safety  burner,  159 
sterilizer,  88 


LABORATORY  outfit,  669-674 
Lactose  litmus,-agar,  142 
Landois,  312 
Leeuwenhoek,  17,  19 
Lens,  oil-immersion,  208 
Leprosy,  bacillus  of,  448 
Leptospira,    ictero-hemorrhagiae, 
631 

icteroides,  631 
Letzerich,  27 
Liebig,  28 
Litmus  gelatin  of  Wurtz,  142 

milk,  growth  of  bacteria  in,  138 
Litmus- whey  milk,  140 
Loffler,  32 

Loffler's    alkaline    methylene-blue 
solution,  178 

method  of  staining  flagella,  187 

serum  mixture,  143 
Lukomsky,  27 
Lysin,  299 


682 


INDEX 


M 


MALIGNANT  edema,  bacillus  of,  616. 

See  Bacillus  edema tis. 
Mallein,  478 
Media,  culture,  111-144 
agar-agar,  129 
blood-serum,  133 

from  small  animals,  132 
preservation  of,  138 
bouillon,  124 
Dunham's  peptone  solution, 

140 
gelatin,  125 

-agar  mixture,  144 
lactose-litmus  agar,  142 
litmus-gelatin,  142 
Loffler's  serum  mixture,  143 
milk,  138 

litmus-whey,  140 
neutralization  of,  111-123 
potatoes,  132 
'  reaction  of,  111-123 
changes  in,  215 
determination  of  hydrogen- 
ion  concentration,  115-124 
litmus,  110 
method   of  Barnett    and 

Chapman,  120 
titration,  113 
serum-water,  of  Hiss,  143 
Meningitis,  cerebrospinal,  377 
antiserum  for,  382 
coccus  causing,  377 
Meningococcus,  377.    See    Micro- 
coccus  intracellularis. 
types,  381 

Mercurial  thermoregulator,  161 
Metatrophic  bacteria,  34 
Metchnikoff,  287 
Micrococci,  66 
Micrococcus  aureus,  345 
abscesses  from,  349 
antiserum  for,  353 
characteristics  of,  345-352 
cultural,  346-348 
pathogenic,  348 
inoculation  with,  348 
microscopic  study  of,  359 
source,  345 
toxins,  352 
citreus,  354 


Micrococcus     epidermidis  •    albus, 

354 

gonorrhea,  370-377 
characteristics  of,  376 
cultivation  of,  371-374- 
morphology,  370 
organisms  simulating,  375 
pathogenesis  of,  374 
peculiarities  of,  376 
staining  reactions  of,  371 
intracellularis,  377 
antiserum  for,  382 
cultures  of,  378 
discovery  of,  377 
inoculation  with,  380 
morphology  of,  377 
peculiarities  of,  378 
varieties  of,  381 
lanceolatus,  369 
pyogenic,  345-368 
tetragenus,  421 
Microscope,  parts  of,  204 
Microscopic  examination,  204,  207- 

211 
Microspira  comma,  549-573.     See 

Cholera. 

Metchnikovi,  574-579 
cultivation  of,  575-578 
discovery  of,  574 
immunity  from,  579 
inoculation  experiments,  578 
morphology  of,  574 
pathogenesis  of,  578 
resistance  of,  578 
source  of,  574 
Schuylkilliensis,  580-582 
cultivation  of,  580-581 
morphology  of,  580 
pathogenesis  of,  581 
Miliary  abscesses,  350-352 
Milk  as  culture  medium,  138 

preparations  of,  138 
bacteriological  analysis  of,  666 
microscopic,  666,  668 
qualitative,  667 
quantitative,  667 
Moitessier's  gas  pressure  regulator, 

163 

Molecular  tremor,  210 
Morphology  of  bacteria,  63,  192 
Motility  of  bacteria,  74 
Mouth,  organisms  in,  401 


INDEX 


683 


Moxter,  299 

Multiplication  of  bacteria,  66,  70 


N 


NASSILOFF,  27 

Needham,  22 

Neisser,  370 

Neutralization   of   culture   media. 

See  Reaction. 
Nitrates,  reduction  of,  224 
Nitrifying  bacteria,  38,  600-606 

cultivation  of,  603 

function  of,  600-602 

morphology  of,  603-606 

where  found,  600 
Nitrites,  tests  for,  224 
Nitrogen  fixation,  39,  602 
Nocard  and  Roux,  263 
Nutrition  of  bacteria,  52-55 
NuttalPs  platinum  spear,  257,  293 


OERTEL,  27 
Ogston,  368 
Oil-immersion  lens,  208 

system,  208 
Opsonin,  294 
Orth,  27 

Oven  incubator,  158 
Oxygen,  cultivation  without,  227 

relation  of  bacteria  to,  55 
Ozanam,  21 


FAKES'  counting  apparatus,  657 
Pappenheim's  decolorizer,  183 
Parasitic  bacteria,  34-36 
Paratrophic  bacteria,  34 
Paratyphoid    bacillus,     538.     See 

Bacillus  paratyphosus. 
Passet,  368 
Pasteur,  21,  23,  28,  30 
Pathogenic  properties  of  bacteria, 

266-272 
Peptone,  purity  of,  141 

solution,  140 
Pest,  391 


Petri  dish,  152 
Phagocytosis,  287 
Photogenic  bacteria,  37 
Plague,  391-400 
antiserum,  399 
bacillus,  392.     See  Bacillus  pes- 

tis. 

historic  outbreaks  of,  391 
protective  inoculation,  397 
reports  on,  400 
Plates,  in  isolating  bacteria  in  pure 

culture,  149-153 
Plenciz,  20,  21 

Pleuro-pneumonia  of  cattle,  263 
Pneumococcus,  404 
Pneumonia,  414 
antiserum,  419 
bacterium  of,  404 
cultivation  of,  407 
discovery  of,  406 
immunization,  419 
inoculation  of,  405,  408 
morphology  of,  405,  406 
pathogenesis  of,  409 
types,  410-412  • 
crisis  in,  417 

mechanism  of  infection,  414 
Pollender,  21,  27 

Post-mortem  examination  of  ani- 
mals, 255-259 
Postulates  of  Koch,  440 
Potato  culture,  194,  215 
preparation  of,  132 
Precipitins,  299 
Proteins,  bacterial,  51 
Prototrophic  bacteria,  34 
Pseudodiphtheric  bacteria,  497 
Pseudomonas  seruginosa,  383 
color  variations  in,  384 
cultivation  of,  384-385 
enzymes  of,  388 
experiments,  384,  388 
inoculation  of,  390 
morphology  of,  383 
pathogenesis  of,  390 
Pseudotuberculosis,     bacteria     of, 

457,  468 

Ptomains,  44,  51 
Pure  cultures,  167 
isolation  of,  149 
plates,  149 
serial  tubes,  155      f 


684 


INDEX 


Pyocyaneus      bacillus,    363.     See 

Pseudomonas  seruginosa. 
Pyogenic  bacteria,  common,  345- 

368 
less  common,  368 


REACTION,  adjustment  of,  111-124 

Barnett  and  Chapman,  120 

changes  in,  215 

hydrogen-ion  concentration,  115- 
124 

litmus  as  indicator,  111 

pH  values,  115-124 

titration  method,  113-115 
Receptors,  301 
Recklinghausen,  27 
Reduction  by  bacteria,  223 
Regulators,  pressure,  164 

thermo-,  160-163 
Resistance,  277 
Rhinitis,  membranous,  bacteria  of, 

494 

Rindfleisch,  26 
Rosenbach,  268 
Roux  and  Yersin,  290 


S 


SALIVA,  study  of,  403     . 
Sanderson,  29 
Saprogenic  bacteria,  37 
Saprophytic  bacteria,  34-35. 
specific  functions  of,  36-40 
chrompgenesis,  37 
denitrification,  39 
nitrification,  38 
nitrogen  fixing,  39 
photogenesis,  37 
saprogenesis,  37 
thiogenesis,  38 
zymogenesis,  38 
Sarcina,  68 
tetragena,  421 

cultivation  of,  422 
discovery  of,  421 
inoculation  of,  423 
morphology  of,  421 
Schaefer,  334 


Schizomycetes,  33 

Schroder  and  Dusch,  23 

Schulze,  23 

Sedgwick  and  Tucker,  663 

Septicemia,  sputum,  404 

Serial  tube  method  of  separation, 

155 

Serum,  blood.     See  Blood  serum. 
Serum-water  media  of  Hiss,  143 
Sewage  streptococci,  659 

presence  of,  in  water,  660 
Skin  disinfection,  343 
Smear  cultures,  167 
Smegma  bacillus,  449 
Soil,  bacteria  in,  665 
isolation  of,  665 
Spallanzani,  30 
Species,  types  of,  203 

variations  of,  199 
Spirilla,  67-70 

Spirillum  of  Asiatic  cholera,     549- 

574.     See  Cholera,  bacteria  of. 

Metchnikovi,  574.     See    Micro- 

spira  Metchnikovi. 
Schuylkilliensis,  580.  See  Micro- 

spira  Schuylkilliensis. 
Spirochsetacese,  629-636 
classification  of,  629-630 
definition  of,  629 
detection  of,  632 
in  smears,  632 
in  tissues,  634 
genera  of,  630 
leptospira,  631 

ictero-hemorrhagiae,  631 
icteroides,  631 
spironemia,  630 

in  relapsing  fever,  630 
treponema,  631 
pallada,  631 
pertenue,  631 

Spironema  of  relapsing  fever,  630 
Spores,  discovery  of,  24 
formation  of,  72-74,  211 
methods  of  study,  209-213 
staining  of,  186 
Sputum,  bacteria  in,  401-403 
Stab  cultures,  167 
Staining,  176-190 
cover  slips,  172,  632 
methods,  176 
acetic  acid,  185 


INDEX 


685 


Staining  methods,    aniline   water, 

178 

carbol-fuchsin,  179 
Duckwall's,  189 
flagella,  187 
Gabbett's,  183 
Gram's,  184 
Loffler's,  178 
Neisser's,  500 

of  spirocheta  pallida,  632,  633 
spores,  186 
Stern's,  633 

of  tubercle  bacillus,  181 
solutions,  176-190 
Koch-Ehrlich's,  178 
Loffler's  blue,  178 
Ziehl's  carbol-fuchsin,  179 
Staphylococcus,  67 
aureus,  345.    See    Micrococcus 

aureus. 
citreus,  354 
epidermidis  albus,  354 
Staphylotoxin,  352 
Steam,  sterilization  by,  80-90 

.  experiments,  327-330 
Sterilization,  chemical,  95 
definition  of,  76-78 
experiments  in,  327-330 
heat,  78 
methods,  79 
direct,  85 

discontinued,  80-85 
fractional,  80-85 
hot  air,  91-94 
pressure,  90 
steam,  87-95 
Sterilizers,  88-93 
Sternberg,  339,  406 
Stern's  staining  method,  633 
Streptococci,  67 
Streptococcus  hemolyticus,  360 
pyogenes,  354,  355-368 
antiserum,  367 
cultivation  of,  357-359 
hemolysis  from,  360 
inoculations,  359-361 
isolation  of,  355 
morphology  of,  356 
pathogenesis  of,  359-361 
source  of,  359 
types  of,  363 
variations  and  varieties  of,  361 


Streptococcus    pyogenes    viridans, 

358 

virulence  of,  360-361 
Suppuration,  causes  of,  368-370 
Sweet's  animal  holder,  236 
Symbiosis,  58 
Symptomatic  anthrax,  bacillus  of, 

621 
Syphilis,  diagnosis  of,  319 

treponema  of,  631 


TEST-TUBE  cultures,  167 

preparing  and  filling  of,  146 
Tests  for  ammonia,  225 

for  hydrogen  sulphide,  224 

for  indol,  221 

for  nitrates,  224 

for  nitrites,  224 

for  toxins,  226 
Tetanus,  antitoxin  for,  614 

bacillus  of,  606-615 
Tetrads,  68 

Thermal  death-point,  94 
Thermo  regulators,  160-163 
Thermophilic  bacteria,  57 
Thiogenic  bacteria,  38 
Tiegel,  29,  30 
Toxins,  44,  46,  47,  272 

action  of,  272 

antitoxins,  273 

bacterial,  226,  272 

formation  of,  226 

immunity  from,  277 

intracellular,  274 

nature  of,  272 

reactions  of,  272 

tests  for,  226 
Toxoids,  274 
Toxones,  274 
Treponema  pallada,  631 

pertenue,  631 
Treviranus,  30 
Tubercles,  miliary,  406 
Tuberculin,  446 

Tuberculosis,  bacillus  of,  441-448 
cultivation  of,  441 
isolation  of,  441 
lesions  produced  by,  430-437 
morphology  of,  444 


686 


INDEX 


Tuberculosis,   bacillus    of,   organ- 
isms simulating,  448 
parasitic  tendencies  of,  441 
peculiarities  of,  441,  445 
staining  of,  445 
variations  of,  446 
cassation  in,  433 
cavity  formation  in,  434 
encapsulation  of  foci  in,  436 
giant  cells,  440 
location  of  bacilli  in,  439 
miliary  tubercles,  432 
modes  of  infection  in,  438 
organisms  with  which  it  may  be 

confused,  448 
primary  infection  in,  436 
susceptibility  of  animals  to,  446 
vaccination  against,  447 
Tubes,  Booker,  154 
Esmarch,  153 
fermentation,  218-221 
filling  of,  146 
preparation  of,  145 
serial,  155 
Tyndall,  24 
Types  of  bacteria,  203 
Typhoid  fever,  agglutination  test 

in,  515 
bacillus  of,  508.     See  Bacillus 

typhosus. 
prophylactic    vaccination    in, 

526 

vaccine  in,  preparation  of,  528 
water  and,  519 
Widal's  reaction  in,  515 


UNSTAINED  preparations,  207,  209 


VACCINATION,  bacterial,  281 
Van  Dungeon,  296 
Variation  of  species,  199 
Variations  and  varieties  of  bacteria, 

199 
Vibrio    Metchnikovi,    574.        See 

Microspira   Metchnikovi. 
Schuylkilliensis,  580.  See  Micro- 
spira Schuylkilliensis. 


Vibrion  septique,  616.     See  Bacil- 
lus edematis. 

Viruses,  filtrable,  261 
ultra-microscopic,  261 


W 


WALDEYER,  26 
Wassermann  reaction,  319 
Water  bacteria,  641 
bacteriological  study  of,  637-660 
collection  of  samples  for,  647 
colon  bacteria,  641 
counting  colonies  in,  654 
apparatus,  655-658 
disease  from,  638 
interpretation     of     results, 

635-643 
objects  of,  638 
pathogenic  bacteria  in,  646 
precautions  in,  644 
qualitative,  643 

special  methods,  648 
quantitative,  649 

methods,  649-654 
sewage  in  659 
typhoid  bacteria,  640 
Weichselbaum,  377 
Weigert,  30,  296 
Weigert's     doctrine,     hyperplasia, 

296 

Welchii  bacterium,  627 
Widal's  reaction,  517 
Wilde,  27 
WolffhugeFs   counting   apparatus, 

655 

Wound  infections,  26-27 
Wright  and  Douglass,  293-294 
Wurtz,  litmus  gelatin  of,  142 


XEROSIS  bacillus,  499 

differentiation  of,  500-501 


ZIEHL'S     carbol-fuchsin    solution, 

179 

Zymogenic  bacteria,  38 
Zymophores,  302 


, 


I  VJ 


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LIBRARY 

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UNIVERSITY  OF  CALIFORNIA  LIBRARY 


